Treatment of disorders associated with elevated blood glucose or blood pressure

ABSTRACT

The present invention relates to methods for treating or delaying the onset of pathologies associated with elevated blood glucose and/or elevated blood pressure. The methods are directed to modulating, regulating, or inhibiting the expression or activity of Connective Tissue Growth Factor (CTGF) or fragments thereof.

This application is a continuation of U.S. patent application Ser. No.10/687,479, filed Oct. 16, 2003, which is a continuation of U.S. patentapplication Ser. No. 09/392,024, filed on Sep. 8, 1999; and claims thebenefit of U.S. Provisional Application Ser. No. 60/099,471, filed onSep. 8, 1998; and U.S. Provisional Application Ser. No. 60/112,855,filed on Dec. 16, 1998; all of which are incorporated by referenceherein in their entirety.

FIELD OF THE INVENTION

The present invention relates to the role of Connective Tissue GrowthFactor (CTGF) in the production of extracellular matrix. Morespecifically, the invention relates to methods of detecting, preventing,and treating kidney fibrosis and other conditions associated withoverproduction of the extracellular matrix by targeting CTGF.

BACKGROUND OF THE INVENTION

Kidney Diseases And Disorders. The kidney functions to separate wasteproducts from the blood, regulate acid concentration, and maintain waterbalance. Kidneys control the levels of various compounds in the blood,such as hydrogen, sodium, potassium, and silicon, and eliminate waste inthe form of urine. Any degradation in kidney function can interfere withthe body's ability to adequately remove metabolic products from theblood, and can disrupt the body's electrolyte balance. In its mostsevere forms, degradation or impairment of kidney function can be fatal.

A number of conditions can lead to chronic renal failure, a decline inkidney function over time. For example, such conditions as hypertension,diabetes, congestive heart failure, lupus, and sickle cell anemia havebeen associated with renal failure. Acute disease processes and injuriescan trigger a more immediate decline in kidney function.

It is thus well understood that individuals with diabetes, hypertension,inflammatory and autoimmune diseases, and other disorders are at riskfor altered and progressive loss of kidney function characterized by,for example, reduced glomerular filtration, albuminuria, proteinuria,and progressive renal insufficiency. More than half of the total numberof kidney disorders initiate kidney fibrosis. Fibrosis involves alteredformation or production of fibrous tissue, and can result in theoverproduction and increased deposition of extracellular matrixcomponents.

The extracellular matrix (ECM) is a complex network of variousglycoproteins, polysaccharides, and other macromolecules secreted from acell into extracellular space. The ECM provides a supportive framework,directly influencing various cellular characteristics, including shape,motility, strength, flexibility, and adhesion. In fibrosis,overproduction and increased deposition of ECM materials can result inthickening and malformation of various membranous and cellularcomponents, reducing local flexibility and surface area of the affectedsite, and impairing a number of bodily processes.

Kidney fibrosis is a common pathway in the progression of various formsof renal injury. Kidney fibrosis typically spreads by enlistingpreviously undamaged regions of the kidney. As normal filtrationprocesses decline, function of surviving tissue and of various regionsof the kidney is systematically destroyed. Kidney fibrosis can bemanifested as a diffuse thickening of kidney membranous components, theaccumulation and expansion leading to a loss of filtration surface areaand a corresponding disruption in the body's electrolyte composition andacid-base balance.

Fibrosis of the kidney is observed in a number of conditions, including,for example, diabetic, autoimmune, and transplant nephropathy;hypertension; and certain forms of glomerular injury or disease.Diabetes mellitus (diabetes) is a complex disease that affects severalhundred million people worldwide. Diabetes is characterized byhyperglycemia or elevated levels of glucose in the blood. Glucose cannotenter the body's cells to be utilized and therefore remains in the bloodin high concentrations. When the blood glucose level exceeds thereabsorptive capacity of the renal tubules, glucose is excreted in theurine. Diabetes produces a number of debilitating and life-threateningcomplications.

Progressive nephropathy is one of the most frequent and seriouscomplications of diabetes. See, e.g., Hans-Henrik et al., 1988, DiabeticNephropathy: The Second World Conference on Diabetes Research, NewFrontiers. The Juvenile Diabetes Foundation International, pp. 28-33. Ahallmark of diabetic nephropathy, and of renal sclerosis due to otherforms of renal injury, is early expansion of the glomerular mesangium,largely due to increased accumulation of ECM proteins such as collagentypes I and IV, fibronectin, and laminin. See, e.g., Mauer et al., 1984,J Clin Invest 74:1143-1155; Bruneval et al., 1985, Human Pathol16:477484. This pathological deposition results in impaired filtration,leading to renal failure, a condition requiring transplantation orlife-long dialysis. Current therapies slow but do not arrest or reversethe progressive loss of kidney function. Predominant causal factorsidentified to date also include hyperglycemia, glomerular hypertension,and abnormal cytokine environments. Tuttle, et al., 1991, N Engl J Med324:1626-1632; The Diabetes Control Complications Trial Research Group,1993, N Engl J Med 329:977-986; Hostetter et al., 1981, Kidney Int19:410-415; Anderson et al., 1985, J. Clin. Invest 76;612-619; Border etal., 1993, Am J Kidney Dis 22:105-113.

Hyperglycemia may be damaging, in great part as increased concentrationsof glucose stimulate ECM accumulation by mesangial cells. See, e.g., Ayoet al., 1990, Am. J. Pathol. 136:1339-1348; Heneda et al., 1991,Diabetologia 34:190-200; Nahman et al., 1992, Kidney Int 41:396402;Cortes et al., 1997, Kidney Int. 51:57-68. As shown by Davies et al.,1992, Kidney Intl. 41:671-678, mesangial cells are largely responsiblefor mesangial matrix synthesis in situ. It has further been determinedthat the effect of glucose on mesangial cell matrix production is linkedto increased glucose transport and utilization. Helig et al., 1995, J.Clin. Invest. 96:1802-1814. Moreover, Ziyadeh et al., 1994, J ClinInvest. 93:536-542, have shown the involvement of secreted solublemediators on mesangial cell matrix production.

Renal hypertension, which can appear as a secondary manifestation ofkidney disease in diabetic patients, can also result from other diseasesor disorders, including long-standing hypertension. Secondaryhypertension can be caused by virtually any impairment in renalfunction. A greater understanding of the pathogenic mechanisms forhypertension-induced ECM deposition is developing. For example, indiabetes, an early impairment of normal blood pressure dampening occursat the glomerular afferent arteriole, resulting in the exposure ofglomerular capillaries to large moment-to-moment variations in systemicblood pressure. Hayashi et al., 1992, J Am Soc Nephrol 2:1578-1586;Bidani et al., 1993, Am J Physiol 265:F391-F398. Due to the elasticityof the glomerulus, increased capillary pressure produces expansion ofglomerular structure, resulting in augmentation of the mechanical strainimposed on the mesangial cells. Riser et al., 1992, J Clin Invest90:1932-1943; Kriz et al., Kidney Int Suppl 30:S2-S9. In addition, whencultured mesangial cells are subjected to cyclic strain, the mesangialcells respond by increasing the synthesis and accumulation of collagentypes I and IV, fibronectin, and laminin. Riser et al., 1992, supra.While increased glomerular pressure is common in diabetes, it is notlimited to this disease, and is present in other forms of progressiverenal disorders, including, for example, certain forms of glomerularnephritis and hypertrophy. See, e.g., Cortes et al., 1997, Kidney Int51:57-68.

Kidney fibrosis and associated renal impairment are thus present in theprogression of various diseases and disorders, including diabetes andhypertension, and methods of treating kidney fibrosis are thus greatlydesired.

Transforming Growth Factor β(TGF-β). The few studies conducted to dateregarding the physiological implications of renal disorders anddiseases, and, in particular, those due to diabetes, have focused on therole of transforming growth factor-β (TGF-β) in developing methods fortargeting overproduction (increased synthesis and accumulation) ofextracellular matrix components. The role of cytokine imbalance ininitiating and/or perpetuating glomerular matrix expansion has beenexplored in experimental nephropathy studies involving TGF-β. See, e.g.,Sharma et al., Seminars In Nephrology 1:116-129. Glomerular TGF-βactivity is increased in both human and experimental diabeticglomerulosclerosis. See, e.g., Yamamoto et al., 1993, Proc Natl Acad Sci90:1814-1818; Sharma et al., 1994, Am J Physiol 267:F1094-F1101;Shankland et al., 1994, Kidney Int 46:430-442. The exposure of culturedmesangial cells or glomeruli to TGF-β results in increased ECMproduction. See, e.g., Bollineni et al., 1993, Diabetes 42:1673-1677. Invivo induction of glomerular matrix accumulation following transfectionand overexpression of the TGF-β gene in rat kidney has been demonstratedby, for example, Isaka et al., J Clin Invest 92:2597-2601.

In addition, neutralization studies have shown that anti-TGF-β antibodymitigates the enhanced glomerular ECM gene expression that occurs inexperimental glomerulonephritis and diabetes. Border et al., 1990,Nature 346:371-374; Sharma et al., 1996, Diabetes 45:522-530. Thesustained overexpression of glomerular TGF-β in diabetes may be theresult of a mesangial cellular response to both increased glucose levelsand hypertension. It has been reported that exposure of mesangial cellsto increased concentrations of glucose in the medium stimulates thesynthesis and release of TGF-β1, as well as the increased binding ofTGF-β to specific receptors. Ziyadeh et al., 1994, J Clin Invest93:536-542; Riser et al., 1998, J Am Soc Nephrol 9:827-836; Riser etal., 1999, Kidney Int 56:428439. It has also been reported thatmechanical force selectively stimulates the production, release, andactivation of TGF-β1, as well as the increased expression of TGF-βreceptors. Riser et al., 1996, Am J Path 148:1915-1923.

In vitro neutralization studies of TGF-β demonstrated a significantreduction of collagen synthesis induced in mesangial cells by increasedglucose levels. See, e.g., Sharma et al., 1996, supra; Ziyadeh et al.,1994, supra. Studies have also shown a virtual elimination of collagenaccumulation resulting from cyclic stretching in the presence of excessglucose. Riser et al., 1997, supra. TGF-β stimulates the proliferationof mesangial cells in vitro and in vivo, and may induce in thesereplicating cells overproduction and increased deposition of ECMcharacteristic of various renal disorders, including proliferativedisorders such as glomerular nephritis. See, e.g., Border et al., 1990,Nature 346:371-374; Habershroh et al., 1993, Am J Physiol 264:F199-205.As a result of these findings, intense efforts have been directed towardreducing TGF-β availability and binding as a means of mitigating matrixaccumulation. However, the ubiquitous nature and pluripotent functionsof TGF-β, including tumor suppression and the multiple levels ofregulation, raise questions concerning both the feasibility and thesafety of its long-term inhibition. See, e.g., Brattain et al., 1996,Curr Opin Oncol 8:49-53; Franklin, 1997, Int J Biochem Cell Biol29:79-89.

Therefore, a method for treating or preventing ECM overproduction orincreased deposition, without interfering with the ubiquitous functionof TGF-β, is needed.

Connective Tissue Growth Factor (CTGF). CTGF is a peptide that may actdownstream of TGF-β to regulate matrix accumulation. This novel growthfactor has been reported and described previously. See, e.g., U.S. Pat.No. 5,408,040; Bradham et al., 1991, J Cell Biol 114:1285-1294. CTGF ischaracterized as a polypeptide which exists as a monomer with amolecular weight of approximately 36 to 38 kD. CTGF has been shown to beone of seven cysteine-rich secreted proteins belonging to the CCNfamily, which includes CTGF, cyr-61, and nov. Oemar et al., 1997,Arterioscler Thromb Vasc Biol 17(8):1483-1489. CTGF is an immediateearly response gene that codes for a protein consisting of four modulesand one signal peptide. Oemar et al., 1997, supra. The four modulesare: 1) an insulin-like growth factor (IGF) binding domain, 2) a vonWillebrand factor type C repeat most likely involved in oligomerization,3) a thrombospondin type 1 repeat believed to be involved in binding tothe ECM, and 4) a C-terminal module which may be involved in receptorbinding. Recent reports suggest that certain fragments of the whole CTGFprotein possess CTGF activity. See, e.g., Brigstock, et al., 1997, JBiol Chem 272(32):20275-20282. Human, mouse, and rat CTGF are highlyconserved with greater than 90% amino acid homology and a molecularweight of about 38 kD. It was recently shown that the promoter of CTGFcontains a novel TGF-β responsive element. Grotendorst et al., 1996,Cell Growth Differ 7:469-480.

It appears that CTGF may be an important prosclerotic molecule in bothskin fibrosis and cardiac atherosclerosis. For example, CTGF mRNA isexpressed by fibroblasts in the lesions of patients with systemicsclerosis, keloids, and localized scleroderma, while there is nocoresponding expression in adjacent normal skin. See, e.g., Igarashi etal., 1995, J Invest Dermatol 105:280-284; Igarashi et al., 1996, JInvest Dermatol 106:729-733. Cultured normal human skin fibroblastsrespond to TGF-β but not to platelet-derived growth factor (PDGF),epidermal growth factor (EGF), or basic fibroblast growth factor (bFGF),by increasing levels of CTGF mRNA and CTGF protein. Igarashi et al.,1993, Mol Biol Cell 4:637-645. Fibroblasts from lesions of sclerodermashow increased mitogenesis to TGF-β and produce greater amounts of CTGFthan do normal fibroblasts. Kikuche et al., 1995, J Invest Dermatol105:128-132. Recombinant human CTGF injected under the skin of NIH Swissmice induces the same rapid and dramatic increase in connective tissuecells and ECM as occurs with TGF-β treatment, whereas PDGF and EGF havelittle or no effect on granulation. Frazier et al., 1996, J InvestDermatol 107:404-411. Cultured vascular smooth muscle cells are alsostimulated by TGF-β to produce CTGF. In heart disease patients, CTGFmRNA is expressed at levels 50- to 100-fold higher in atheroscleroticplaques than in normal arteries. Oemar et al., 1997, Circulation95(4):831-839.

In spite of mounting evidence implicating CTGF as a causal factor inskin fibrosis and cardiac atherosclerosis, very little is known of itsexpression in, for example, renal sclerosis or diabetes. It has beenshown, using an in vitro model of calcium oxalate nephrolithasis, thatmonkey kidney epithelial cells respond to calcium oxalate byupregulating the CTGF gene along with other genes involved in matrixturnover. Hammes et al., 1995, Kidney Int 48:501-509. A similar responseoccurs in cultured renal epithelial cells following mechanical wounding.See, e.g., Pawar et al., 1995, J Cell Physiol 165:556-565. Mostrecently, CTGF mRNA was found in biopsies from normal human kidneys. Aqualitative assessment indicated that, in a limited number of cases,CTGF expression was increased in the tissues of patients with severemesangial proliferative lesions of crescentic glomerulonephritis, focaland segmented glomerulosclerosis, and, in three cases, diabeticglomerulosclerosis. Ito et al., 1998, Kidney Int 53:853-861. Theresearch, relying only on data obtained from biopsies, did not includequantitative results or any measurement of CTGF protein levels. Further,no connection between CTGF mRNA levels and the production and depositionof ECM, and no quantitative method for detecting renal disorders ordiseases, including diabetes, involving a determination of CTGF levelsin samples, and did not identify CTGF-expressing cells.

The role of CTGF in kidney diseases is thus unclear, and there has beenno research to date has shown that CTGF is causally related to ECMoverproduction and increased deposition and to fibrosis in the kidney.

Diagnostics and Early-Stage Detection. Kidney failure is a seriouscondition requiring extreme treatment such as hemodialysis ortransplantation. Early-stage detection and/or prevention of anydeviation from normal kidney pathology and function could minimize therisk of a subject's developing a more serious condition. Hypertension,for example, might be undetectable by a patient in early stages, but canbe deadly if not identified, monitored, and treated. In addition, insome diseases, such as, for example, diabetes, less invasive anddisruptive and more affordable means of treatment, such as dietarymodification, are effective only at early stages. Therefore, there is acritical need for effective and reliable methods of diagnosis thatpermit early stage detection, and corresponding prevention, of renalcomplications.

For example, kidney failure resulting from progressiveglomerulosclerosis is the leading cause of morbidity and mortality amongpatients with type I, or juvenile, diabetes mellitus. See, e.g., Dormanet al., 1984, Diabetes 33:271-276; Anderson et al., 1983, Diabetologia25:496-501. Current therapy with angiotensin-converting enzyme (ACE)inhibitors, the drug class of choice, effectively slows the progressionof disease. See, e.g., Lewis et al., 1993, N Eng J Med 329:1456-1462.Nevertheless, this treatment is not justified in all newly diagnoseddiabetic patients because only approximately 30-35% of these developprogressive kidney disease, and the long-term side effects of thesedrugs are uncertain. See, e.g., Parving and Hommel, 1989, Brit Med J299:230-233. In addition, ACE inhibitors are also presently used totreat patients with hypertensive renal failure, including that resultingfrom non-diabetic nephropathies. However, the mechanism of renalprotection, and, as noted above, the long-term side effects of thistreatment are not fully understood. Furthermore, ACE inhibitors havebeen shown to negatively interact with nonsteroidal anti-inflammatorydrugs. See, e.g., Whelton, 1999, Am J Med 106(SB):13S-24S.

In a current method of diagnosis, diabetic patients are monitored formicroalbuminuria. Persistent microalbuminuria is a marker of widespreadvascular damage and indicates the presence of early nephropathy in type1 and type 2 diabetes. See, e.g., Stehouwer et al., 1992, Lancet340:319-323; Bojestig et al., 1996, Diabetes Care 19:313-317; Mogensenet al., 1995, Lancet 346:1080-1084. However, the actual level ofmicroalbuminuria may not necessarily predict the development of overtnephropathy, particularly among patients with a long duration ofdiabetes. Bojestig et al., supra. In addition, since by the timemicroalbuminuria is detected, structural renal lesions are alreadypresent, the effectiveness of treatment to slow progression may besubstantially reduced. Bangstad et al., 1993, Diabetologia 36:523-529;Ruggenenti et al., 1998, J Am Soc Nephrol 9:2157-2169; Fioretto et al.,1995, Kidney Int 48:1929-1935. There is a great need to be able topredict which patients with type 1 diabetes will develop nephropathy,and to, in general, develop a method that will detect renal alterationsthat may precede the onset of significant disease.

In summary, there is a need in the art for effective methods fordiagnosing, treating, and preventing fibrosis associated with impairmentand degradation of kidney function in a variety of diseases anddisorders, most particularly, in diabetes and hypertension. No currentresearch has focused on the modulation of CTGF expression or activity asa means of preventing or treating kidney fibrosis.

SUMMARY OF THE INVENTION

The present invention satisfies the need in the art by providing methodsfor detecting, treating, and preventing renal disorders and diseasesassociated with fibrosis. In particular, the present invention providesmethods for detecting, preventing, and treating pathologies andcomplications associated with renal disorders and conditions which arecharacterized by an overproduction or increased deposition ofextracellular matrix.

Methods Of Treatment and Prevention. The present invention providesvarious approaches directed to modulation of the overproduction of theextracellular matrix resulting in fibrosis. Specifically, the presentinvention provides methods of regulating increased accumulation of theextracellular matrix associated with kidney fibrosis as found in variousrenal diseases and disorders. These renal diseases and disordersinclude, but are not limited to, all kinds of nephropathy, includingglomerulonephritis, glomerulosclerosis, and conditions resulting fromglomerular injury; diabetic nephropathy and other complications;nephritis; interstitial disease; acute and chronic transplant rejection;renal hypertension, including that associated with diabetes; and otherunderlying causes of fibrosis. More specifically, the present inventionprovides methods for preventing and treating complications associatedwith the above-named renal disease and disorders by regulating,modulating, and/or inhibiting the expression and activity of CTGF. Inparticular embodiments, the present methods are directed to thediagnosis, prevention, and treatment of renal diseases and disordersassociated with diabetes or with hypertension.

The methods of the present invention provide for the administration of atherapeutically effective amount of an agent that regulates, modulates,and/or inhibits the ECM-producing activity of CTGF. In particular, themethods of this invention are useful for the treatment and prevention ofrenal disorders in mammals, most preferably, in humans.

In one aspect, the invention provides a method of treating complicationsassociated with diabetes characterized by the overproduction oroveraccumulation of the extracellular matrix by administering atherapeutically effective amount of an antibody reactive with a CTGFpolypeptide or fragments thereof, or an antigen-binding fragment of anantibody reactive with the CTGF polypeptide or fragments thereof.

In another aspect, the present invention provides a method for treatingand preventing complications associated with renal disorders,particularly, diabetes and hypertension, wherein antisenseoligonucleotides which specifically bind to CTGF mRNA are used tointerrupt expression of the protein product. The antisenseoligonucleotides have a sequence capable of binding specifically withany polynucleotide sequences encoding CTGF or fragments thereof.

In yet a further embodiment of the present invention, a method isprovided in which small molecules are used to inhibit the activity ofCTGF or its active fragments by blocking the binding of CTGF to itsreceptor, inhibiting CTGF activity and thus thereby reducing theoverproduction of the extracellular matrix associated with the onsetand/or progression of renal disorders, including diabetes andhypertension.

The present invention further provides a method of treating andpreventing renal disorders by administering a compound that blocks thebinding interactions of or the enzymes involved in the signaltransduction pathway of CTGF.

The present invention also provides a method for treating and preventingdiabetes by administering insulin and an agent that modulates and/orinhibits the activity of CTGF. More specifically, the present inventiondiscloses a method for treating and preventing diabetes by administeringinsulin and an agent that modulates, regulates, and/or inhibits theactivity of CTGF according to the methods of the present invention.

Methods for evaluating the effectiveness of anti-fibrotic therapy,including the use of ACE inhibitors, by measuring the levels of CTGF ina sample from a subject undergoing a course of treatment for diseasesand disorders associated with fibrosis, are also provided.

Diagnostic Methods. The present invention is also directed to methodsfor predicting which patients with diseases and disorders associatedwith renal disorders will subsequently develop progressive kidneydisease. In one embodiment, the invention provides a method fordetecting and/or staging (classifying the level, site, and spread ofdisease) kidney involvement in a particular disease or disorder. In oneembodiment, a method of predicting whether a patient with diabetes willgo on to develop progressive kidney disease is provided, along with amethod of detecting the current level of kidney involvement, forexample, in a subject with diabetes as opposed to a subject withoutdiabetes.

The present invention is also directed to methods of detecting thepresence of pathology of a tissue characterized by an excessiveaccumulation of extracellular matrix components. In one embodiment, themethod involves determining the levels of CTGF, for example, throughtissue biopsy or through non-intrusive methods, such as, for example,collection of a urine sample. In a particular embodiment, the methodcomprises determining the levels of CTGF in a sample comprising, forexample, urine or other bodily fluids from a subject with a diabeticnephropathy, such as, for example, diabetic glomerulosclerosis. Themethod can also comprise determining the levels of CTGF in persons withprogressive sclerosis, both with and without diabetes, by determiningthe levels of CTGF in urine or in other bodily fluids.

More specifically, the present invention comprises a means of diagnosingthe presence of or a predisposition to kidney diseases and disorders,including a means for detecting and monitoring the pathogenesis of thesediseases and disorders, or for detecting and monitoring the presence ofmarkers for the pathogenesis of these diseases and disorders. Morespecifically, the present invention provides for diagnosing renaldisorders by measuring the levels of CTGF in a patient sample,preferably, in a urine sample from a patient.

In one embodiment of the present invention, a method is provided for themeasurement of CTGF levels in a sample from a patient with no known orwith a suspected renal disorder. Comparison of CTGF levels in urinesamples from a patient known to have a kidney disease or disorder, orfrom a patient known not to have any kidney disease or disorder, withCTGF levels in urine samples from a patient with no known or with asuspected kidney disorder can be indicative of the presence of a kidneydisease or disorder. In particular, the method provides that higherlevels of CTGF are present in samples from patients having renaldisorders than in samples from patients without any renal disorder.Higher levels of CTGF are thus indicative of the presence of a diseaseor disorder associated with kidney fibrosis.

In another embodiment, the levels of CTGF can be measured by detectingCTGF mRNA or protein in a sample. In a further embodiment, the sample isa tissue sample and the presence of CTGF is detected by staining of theprotein in the tissue or by determination of CTGF mRNA levels.

A preferred method of the present invention utilizes an antibody,preferably, a monocolonal antibody, capable of specifically binding toCTGF or active fragments thereof. The method of utilizing an antibody tomeasure the levels of CTGF allows for non-invasive diagnosis of thepathological states of kidney diseases. In a preferred embodiment of thepresent invention, the antibody is human or is humanized. The preferredantibodies may be used, for example, in standard radioimmunoassays orenzyme-linked immunosorbent assays or other assays which utilizeantibodies for measurement of levels of CTGF in sample. In a particularembodiment, the antibodies of the present invention are used to detectand to measure the levels of CTGF present in a urine sample.

Diagnostic Kit. The present invention is further directed to diagnostickits for detecting and measuring the levels of CTGF in a sample in orderto detect a renal disorder or a predisposition to a renal disorder in asubject. In one embodiment, the kit contains antibodies specific forCTGF and reagents for detecting and measuring CTGF in a sample. Thesample can be a bodily fluid, such as urine, or, for example, a tissuesample. In one embodiment of the present invention, the kit comprises animmobilized antibody which specifically recognizes CTGF and an antibodyspecific for CTGF and capable of binding to an antigen componentdifferent from the immobilized antibody. The CTGF antibody can beenzyme-labeled, radio-labeled, or fluoroscein-labeled. The kit can alsocomprises reagents necessary for detection of the antibody, and canfurther comprise other reagents as desired, such as, for example,dissolving agents, cleaning agents, and reaction terminators.

In a preferred embodiment of the invention, the kit is packaged, forexample, in a box or a container which includes the necessary elementsof the kit, and also includes instructions relating to the use of thekit.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and FIG. 1B depict the effects of exogenous CTGF on mesangialcell secretion of the extracellular matrix. FIG. 1A shows the quantitiesof fibronectin contained in the media at the end of incubationdetermined by ELISA. FIG. 1B shows the quantities of collagen type Icontained in the media at the end of incubation determined by ELISA.

FIG. 2 depicts CTGF gene expression in rat tissues and cultured kidneycells. Total RNA was extracted from whole organs of rat and fromcultured rat mesangial cells and kidney fibroblasts for Northernanalysis. MC represents mesangial cells; BT represents brain tissue; HTrepresents heart tissue; KT represents kidney tissue; and KFC representskidney fibroblast cells.

FIG. 3A, FIG. 3B, and FIG. 3C depict regulation of CTGF and TGF-β mRNAlevels by exogenous TGF-β and CTGF. FIG. 3A shows results from arepresentative experiment. FIG. 3B shows quantitation of mRNA bands forCTGF. FIG. 3C shows quantitation of mRNA bands for TGF-β.

FIG. 4A and FIG. 4B depict expression of CTGF protein by culturemesangial cells in the presence of exogenous TGF-β. FIG. 4A showsimmunoblotting using an antibody raised against full length CTGF. FIG.4B shows immunoblotting using an antibody raised against a 15 amino acidsequence specific to CTGF.

FIG. 5A and FIG. 5B depict secretion of CTGF protein into the medium ofmesangial cell cultures, and the effect of heparin. FIG. 5A shows datarelating to media pooled and heparin-sepharose treated forimmunoblotting. FIG. 5B shows data relating to media tested for CTGFcontent individually by ELISA prior to pooling.

FIG. 6A and FIG. 6B depict CTGF protein induction by mesangial cells.FIG. 6A shows data relating to media pooled and heparin-sepharosetreated for immunoblotting. FIG. 6A shows data relating to media testedfor CTGF content individually by ELISA prior to pooling.

FIG. 7 depicts the effect of high glucose concentration on mesangialcells expression of CTGF mRNA. FIG. 7 shows samples of pooled RNA from 6different 100 mm culture dishes in a representative experiment.

FIG. 8 depicts TGF-β blockade of high glucose-induced CTGF productionusing anti-TGF-β antibody.

FIG. 9 depicts CTGF concentrations in mesangial cells overexpressingdifferent levels of GLUT1. Supernatants from duplicate culture cellstransduced with the GLUT1 gene denoted MCGT1 or a transfection controlLaZ gene (MCLaZ).

FIG. 10 depicts the effect of cyclic stretching on mesangial cellexpression of CTGF transcripts. At the indicated periods, RNA wasextracted and probed for CTGF message. Each lane represents the resultsof the samples pooled from 24 different culture wells.

FIG. 11 depicts blockade of stimulated collagen type I production by andanti-CTGF antibody.

FIG. 12 depicts blockade of stimulated mesangial cell proliferation byanti-CTGF antibody.

FIG. 13A and FIG. 13B depict glomerular disease associated with diabetesin db/db mice. FIG. 13A shows renal cortical section from control db/mmice at 5 months of age. FIG. 13B shows renal cortical section fromdiabetic db/db mice at 5 months of age. The sections from FIG. 13A andFIG. 13B were stained with PAS for light microscopy examination, and areexamples of glomeruli demonstrating the most severe mesangial expansionobserved in the diabetic group.

FIG. 14A, FIG. 14B, and FIG. 14C depict induction of CTGF andfibronectin transcripts in whole kidney of diabetic db/db mice at 5months of age. FIGS. 14A and 14B show total RNA extracted from wholekidneys and probed by northern analysis for CTGF mRNA and fibronectinmRNA, respectively. The letter “C” represents nondiabetic mice, whilethe letter “D” represents diabetic mice. FIG. 4C shows quantification bydenositometric analysis of the results of the Northern analyses.

FIG. 15A and FIG. 15B depict competitive RT-PCR for GAPDH and CTGF mRNAin a single sample from diabetic mouse glomeruli. Ethidiumbromide-stained gel after PCR amplification. The lanes of FIGS. 11A and11B contain a constant amount of test cDNA and 2-fold decreasingconcentrations of a known amount of the specific mimic. FIG. 15A showscompetitive reverse transcriptase PCR (RT-PCR) for GAPDH. FIG. 15B showscompetitive RT-PCR for CTGF.

FIG. 16 depicts the effects of diabetes on the glomerular expression ofCTGF and fibronectin transcript levels in db/db mice, detected bycompetitive RT-PCR.

FIG. 17 depicts CTGF and its recovery in normal urine. Four aliquotswere spiked with a different amount of CTGF, and a fifth served as acontrol. Immunoblotting was performed using a CTGF specific antibody.

FIG. 18 depicts the analysis of CTGF protein in urine of diseasedpatients or healthy volunteers. Urine samples from 8 patients withkidney disease or 3 normal volunteers were assayed for CTGF byimmunoblotting.

DETAILED DESCRIPTION OF THE INVENTION

It is understood that the present invention is not limited to theparticular methodologies, protocols, cell lines, and reagents, etc.,described herein, as these may vary. It is also to be understood thatthe terminology used herein is used for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention. It must be noted that as used herein and in theclaims, the singular forms “a,” “an,” and “the” include pluralreferences unless the context clearly dictates otherwise. Thus, forexample, a reference to “an antibody” is a reference to one or moreantibodies and any equivalents thereof known to those skilled in theart.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Preferred methods, devices,and materials are described, although any similar or equivalent methodscan be used in the practice or testing of the present invention. Allpatents, publications, and other references cited herein areincorporated by reference herein in their entirety.

Definitions

As used herein, the term “extracellular matrix” refers broadly tonon-cellular matrix, typically composed of proteins, glycoproteins,complex carbohydrates, and other macromolecules. Extracellular matrixcomponents include, for example, collagen types I and IV, fibronectin,laminin, and thrombospondin.

The term “fibrosis” refers to abnormal processing of fibrous tissue, orfibroid or fibrous degeneration. Fibrosis can result from variousinjuries or diseases, and can often result from chronic transplantrejection relating to the transplantation of various organs. Fibrosistypically involves the abnormal production, accumulation, or depositionof extracellular matrix components, including overproduction andincreased deposition of, for example, collagen and fibronectin.

As used herein, the terms “kidney fibrosis” or “renal fibrosis” or“fibrosis of the kidney” refer to diseases or disorders associated withthe overproduction or abnormal deposition of extracellular matrixcomponents, particularly collagen, leading to the degradation orimpairment of kidney function. The terms “disorders” and “diseases” areused inclusively and refer to any condition deviating from normal.“Diseases” and “disorders” include, but are not limited to, allograftand transplant rejection, acute and chronic, and any transplantnephropathy; acute and chronic kidney failure; autoimmune nephropathy;diabetic nephropathy; glomerulonephritis, glomerulosclerosis, and otherforms of glomerular abnormality or injury; hypertension; hypertrophy;interstitial disease; nephritis; sclerosis, an induration or hardeningof tissues and/or vessels resulting from causes that include, forexample, inflammation due to disease or injury; renal-associatedproliferative disorders; and other primary or secondary nephrogenicconditions. Fibrosis associated with dialysis following kidney failureand catheter placement, e.g., peritoneal and vascular access fibrosis,is also included.

It is understood that, while kidney fibrosis is the model for discussionof the present invention, the mechanism of fibrosis is universal.Therefore, the presently described methods, kits, and other aspects ofthe present invention could also be directed to the diagnosis,prevention, and treatment of other forms of fibrosis and diseases anddisorders associated with fibrosis and proliferation, including, but notlimited to: cardiac fibrosis, pulmonary fibrosis, diabetic retinopathy,skin fibrosis, scleroderma, atherosclerosis, arteriosclerosis,hypertropic scarring, keloid formation, arthritis, liver fibrosis,inflammation, tumor growth metastasis, other conditions related to cellproliferation and migration, including those associated with vascularendothelial cells, for example, angiogenesis and neovascularization,etc.

The term “sample” is used herein in its broadest sense. Samples may bederived from any source, for example, from bodily fluids, secretions, ortissues including, but not limited to, saliva, blood, urine, and organtissue (e.g., biopsied tissue); from chromosomes, organelles, or othermembranes isolated from a cell; from genomic DNA, cDNA, RNA, mRNA, etc.;and from cleared cells or tissues, or blots or imprints from such cellsor tissues. A sample can be in solution or can be, for example, fixed orbound to a substrate. A sample can refer to any material suitable fortesting for the presence of CTGF or suitable for screening for moleculesthat bind to CTGF or fragments thereof. Methods for obtaining suchsamples are within the level of skill in the art.

An “antisense sequence” is any sequence capable of specificallyhybridizing to a target sequence. The antisense sequence can be DNA,RNA, or any nucleic acid mimic or analog. The term “antisensetechnology” refers to any technology which relies on the specifichybridization of an antisense sequence to a target sequence.

The terms “modulation” and “regulation” as used with respect to CTGFexpression or activity refer to any direction of or effect on CTGFexpression or activity as compared to normal or to unaltered CTGFexpression or activity.

Invention

A. CTGF and its Role in Fibrosis and Renal Disorders

The present invention is based on the discovery that CTGF is animportant mediator of extracellular accumulation in fibrotic conditions,and, in particular, in fibrotic conditions associated with renaldisorders, such as diabetes and glomerular hypertension. Morespecifically, the present invention is based on the discovery that theproduction of CTGF by glomerular cells (in particular, mesangial cells)is a potentially important factor in the pathogenesis of diseases anddisorders of the kidney. It was discovered that increased levels of CTGFinduced increased production and deposition of ECM in mesangial cells.It was further found in an analysis of urine samples that healthysubjects had no or very minimal levels of CTGF in their urine, while theurine of diabetic patients or patients with other renal disorders showedincreased levels of CTGF.

To demonstrate that CTGF is a critical determinant of extracellularmatrix deposition in the kidney, CTGF expression in mesangial cells,glomeruli, and whole kidney was examined under diabetic and non-diabeticconditions. Mesangial cells cultured in media containing normal levelsof glucose expressed low levels of CTGF mRNA and secreted barelydetectable amounts of the full length CTGF protein. However, in ahyperglycemic environment in which mesangial cells were exposed toelevated glucose levels, upregulation of CTGF expression and increasedprotein production were detected. Moreover, mechanical strain ofmesangial cells, exhibitive of, for example, glomerulosclerosis,glomerular hypertension, and glomerular hypertrophy, demonstrated anupregulated expression and protein production of CTGF in mesangialcells. Thus, the present invention demonstrates that exposure toconditions such as increased glucose concentrations, mechanical force,or TGF-β led to upregulated expression and protein production of CTGF,establishing a nexus between the presence of CTGF and renal disorders,in particular, diabetes and hypertension. Even in the absence ofhypertension, conditions that produce, for example, glomerularhypertrophy, often a result of renal injury, can result in induceincreased capillary vessel diameter. According to Laplace's law, vesselwall tension is correspondingly increased, and increased mesangial cellstretching forces are likely produced. See Cortes et al., 1997, supra.

B. Methods for Modulating and Inhibiting Activity of CTGF

Connective Tissue Growth Factor (CTGF) is a critical determinant ofextracellular matrix deposition in kidney fibrotic conditions. Thepresent invention provides methods for the diagnosis, prevention, andtreatment of complications associated with kidney fibrosis, preferably,by regulating, modulating, and/or inhibiting the expression or activityof CTGF. More specifically, methods of the present invention provide forthe administration of a therapeutically effective amount of an agentthat regulates, modulates, and/or inhibits the extracellular matrixproducing activity of CTGF.

Antibodies. In one embodiment of the present invention, methods fordiagnosis, prevention, and treatment of renal disorders and diseasesinvolve the administration of a therapeutically effective amount of anantibody which specifically reacts with a CTGF polypeptide or fragmentsthereof.

CTGF antibodies may be generated using methods well known in the art.Such antibodies may include, but are not limited to, polyclonal,monoclonal, chimeric, single chain antibodies, as well as Fab fragments,including F(ab′)₂ and F_(v) fragments. Fragments can be produced, forexample, by a Fab expression library. Neutralizing antibodies, i.e.,those which inhibit dimer formation, are especially preferred fortherapeutic use.

A target polypeptide, such as CTGF or an agent that modulates theactivity and or expression of CTGF, can be evaluated to determineregions of high immunogenicity. Methods of analysis and epitopeselection are well known in the art. See, e.g., Ausubel et al., eds.,1988, Current Protocols in Molecular Biology, John Wiley & Sons, Inc.,New York N.Y. Analysis and selection can also be accomplished, forexample, by various software packages, such as LASERGENE NAVIGATORsoftware. (DNASTAR; Madison Wis.) The peptides or fragments used toinduce antibodies should be antigenic, but are not necessarilybiologically active. Preferably, an antigenic fragment or peptide is atleast 5 amino acids in length, more preferably, at least 10 amino acidsin length, and most preferably, at least 15 amino acids in length. It ispreferable that the antibody-inducing fragment or peptide is identicalto at least a portion of the amino acid sequence of the targetpolypeptide, e.g., CTGF. A peptide or fragment that mimics at least aportion of the sequence of the naturally occurring target polypeptidecan also be fused with another protein, e.g., keyhole limpet hemocyanin(KLH), and antibodies can be produced against the chimeric molecule.

Methods for the production of antibodies are well known in the art. Forexample, various hosts, including goats, rabbits, rats, mice, humans,and others, may be immunized by injection with the target polypeptide orany immunogenic fragment or peptide thereof. Depending on the hostspecies, various adjuvants may be used to increase immunologicalresponse. Such adjuvants include, but are not limited to, Freund'sadjuvant, mineral gels such as aluminum hydroxide, and surface-activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, KLH, and dinitrophenol. Among adjuvants used in humans,BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are especiallypreferable.

Monoclonal and polycolonal antibodies may be prepared using anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. Techniques for in vivo and in vitroproduction are well known in the art. See, e.g., Pound, 1998,Immunochemical Protocols, Humana Press, Totowa N.J.; Harlow and Lane,1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory,New York N.Y. The production of chimeric antibodies is also well known,as is the production of single-chain antibodies. See, e.g., Morrison etal., 1984, Proc Natl Acad Sci 81:6851-6855; Neuberger et al., 1984,Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454. Antibodieswith related specificity, but of distinct idiotypic composition, may begenerated, for example, by chain shuffling from random combinatorialimmunoglobin libraries. See, e.g., Burton, 1991, Proc Natl Acad Sci88:11120-11123.

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents. See, e.g., Orlandi et al., 1989,Proc Natl Acad Sci 86:3833-3837; Winter and Milstein, 1991, Nature349:293-299). Antibody fragments which contain specific binding sitesfor the target polypeptide may also be generated. Such antibodyfragments include, but are not limited to, F(ab′)₂ fragments, which canbe produced by pepsin digestion of the antibody molecule, and Fabfragments, which can be generated by reducing the disulfide bridges ofthe F(ab′)₂ fragments. Alternatively, Fab expression libraries may beconstructed to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity. See, e.g., Huse et al., 1989,Science 254:1275-1281.

Antibodies can be tested for anti-target polypeptide activity using avariety of methods well known in the art. Various techniques may be usedfor screening to identify antibodies having the desired specificity,including various immunoassays, such as enzyme-linked immunosorbentassays (ELISAs), including direct and ligand-capture ELISAs,radioimmunoassays (RIAs), immunoblotting, and fluorescent activated cellsorting (FACS). Numerous protocols for competitive binding orimmunoradiometric assays, using either polyclonal or monoclonalantibodies with established specificities, are well known in the art.See, e.g., Harlow and Lane. Such immunoassays typically involve themeasurement of complex formation between the target polypeptide and aspecific antibody. A two-site, monoclonal-based immunoassay utilizingmonoclonal antibodies reactive to two non-interfering epitopes on thetarget polypeptide is preferred, but other assays, such as a competitivebinding assay, may also be employed. See, e.g., Maddox et al, 1983, JExp Med 158:1211.

Antibodies as described above could also be used to identify CTGF orfragments thereof in tissue, e.g., from a kidney biopsy. The amount ofCTGF present could be determined, for example, by quantitative imageanalysis. CTGF mRNA levels could also be determined, such as by reversetranscriptase polymerase chain reaction (PCR) using portions of thebiopsied tissue, e.g., glomeruli. In particular, in this method, mRNAfrom a tissue sample, in total, or that specific for CTGF or fragmentsthereof, could be transcribed to DNA and then amplified through PCRusing CTGF-specific primer sequences. Quantitation of mRNA for CTGF orfragments thereof could be determined, for example, by a competitionreaction using equal volumes of the patient sample run against a seriesof decreasing known concentrations, e.g., of a mimic or mutant cDNAfragment.

The present invention contemplates the use of antibodies specificallyreactive with a CTGF polypeptide or fragments thereof which neutralizethe biological activity of CTGF. The antibody administered in the methodcan be the intact antibody or antigen binding fragments thereof, such asFab, F(ab′)₂, and F_(v) fragments, which are capable of binding theepitopic determinant. The antibodies used in the method can bepolyclonal or, more preferably, monoclonal antibodies. Monoclonalantibodies with different epitopic specificities are made from antigencontaining fragments of the protein by methods well known in the art.See, e.g., Kohler et al., 1975, Nature 256:495-497; Ausubel, et al.,supra.

In the present invention, therapeutic applications include those using“human” or “humanized” antibodies directed to CTGF or fragments thereof.Humanized antibodies are antibodies, or antibody fragments, that havethe same binding specificity as a parent antibody, (i.e., typically ofmouse origin) and increased human characteristics. Humanized antibodiesmay be obtained, for example, by chain shuffling or by using phagedisplay technology. For example, a polypeptide comprising a heavy orlight chain variable domain of a non-human antibody specific for a CTGFis combined with a repertoire of human complementary (light or heavy)chain variable domains. Hybrid pairings specific for the antigen ofinterest are selected. Human chains from the selected pairings may thenbe combined with a repertoire of human complementary variable domains(heavy or light) and humanized antibody polypeptide dimers can beselected for binding specificity for an antigen. Techniques describedfor generation of humanized antibodies that can be used in the method ofthe present invention are disclosed in, for example, U.S. Pat. Nos.5,565,332; 5,585,089; 5,694,761; and 5,693,762. Furthermore, techniquesdescribed for the production of human antibodies in transgenic mice aredescribed in, for example, U.S. Pat. Nos. 5,545,806 and 5,569,825.

In another embodiment of the present invention, a method involves theadministration of a therapeutically effective amount of an antibodyreactive to a CTGF responsive receptor, and, more specifically, anantibody which blocks the binding of CTGF to its cellular receptors. Themethod of the present invention provides that the antibody reactive withCTGF modulates and/or inhibits the biological activity of CTGF throughthe manipulation and control of the interaction between CTGF and itsreceptor by inactivation of the receptor independently of CTGF.

Antisense Oligonucleotides. The present invention provides for atherapeutic approach which directly interferes with CTGF expression.Specifically, a therapeutic approach which directly interrupts thetranslation of CTGF mRNA into protein could be used to bind to CTGF mRNAor to otherwise interfere with CTGF expression. Antisense technologyrelies on the modulation of expression of a target protein through thespecific binding of an antisense sequence to a target sequence encodingthe target protein or directing its expression. See, e.g., Agrawal, ed.,1996, Antisense Therapeutics, Humana Press, Inc., Totowa N.J.; Alama etal., 1997, Pharmacol Res 36(3):171-178; Crooke, 1997, Adv Pharmacol40:1-449; and Lavrosky et al.,1997, Biochem Mol Med 62(1):11-22.Antisense sequences are nucleic acid sequences capable of specificallyhybridizing to at least a portion of a target sequence. Antisensesequences can bind to cellular mRNA or genomic DNA, blocking translationor transcription and thus interfering with expression of a targetedprotein product. Antisense sequences can be any nucleic acid material,including DNA, RNA, or any nucleic acid mimics or analogs. See, e.g.,Rossi et al., 1991 Antisense Res Dev 1(3):285-288; Pardridge et al.,1995, Proc Natl Acad Sci 92(12):5592-5596; Nielsen and Haaima, 1997,Chem Soc Rev 96:73-78; and Lee et al., 1998, Biochemistry37(3):900-1010. Delivery of antisense sequences can be accomplished in avariety of ways, such as through intracellular delivery using anexpression vector. See discussion, infra. Site-specific delivery ofexogenous genes is also contemplated, such as techniques in which cellsare first transfected in culture and stable transfectants aresubsequently delivered to the target site. See, e.g., Kitamura et al.,1994, Kidney Int 43:S55-S58.

Antisense oligonucleotides of about 15 to 25 nucleic acid bases aretypically preferred as such are easily synthesized and are capable ofproducing the desired inhibitory effect. Molecular analogs of antisenseoligonucleotide may also be used for this purpose and can have addedadvantages such as stability, distribution, or limited toxicityadvantageous in a pharmaceutical product. In addition, chemicallyreactive groups, such as iron-linked ethylenediamine-tetraacetic acid(EDTA-Fe), can be attached to antisense oligonucleotides, causingcleavage of the RNA at the site of hybridization. These and other usesof antisense methods to inhibit the in vitro translation of genes arewell known in the art. See, e.g., Marcus-Sakura, 1988, Anal Biochem172:289.

Delivery of antisense therapies and the like can be achievedintracellularly through using a recombinant expression vector such as achimeric virus or a colloidal dispersion system which, upontranscription, produces a sequence complementary to at least a portionof the cellular sequence encoding the target protein. See, e.g., Slateret al., 1998, J Allergy Clin Immunol 102(3):469-475. Delivery ofantisense sequences can also be achieved through various viral vectors,including retrovirus and adeno-associated virus vectors. See, e.g.,Miller, 1990, Blood 76:271; and Uckert and Walther,1994, Pharacol Ther63(3):323-347. Vectors which can be utilized for antisense gene therapyas taught herein include, but are not limited to, adenoviruses, herpesviruses, vaccinia, or, preferably, RNA viruses such as retroviruses.

Retroviral vectors are preferably derivatives of murine or avianretrovirus. Retroviral vectors can be made target-specific by inserting,for example, a polynucleotide encoding a protein or proteins such thatthe desired ligand is expressed on the surface of the viral vector. Suchligand may be a glycolipid carbohydrate or protein in nature. Preferredtargeting may also be accomplished by using an antibody to target theretroviral vector. Those of skill in the art will know of, or canreadily ascertain without undue experimentation, specific polynucleotidesequences which can be inserted into the retroviral genome to allowtarget specific delivery of the retroviral vector containing theantisense polynucleotide.

Recombinant retroviruses are typically replication defective, and canrequire assistance in order to produce infectious vector particles. Thisassistance can be provided by, for example, using helper cell lines thatcontain plasmids encoding all-of the structural genes of the retrovirusunder the control of regulatory sequences within the LTR. These plasmidsare missing a nucleotide sequence which enables the packaging mechanismto recognize an RNA transcript for encapsidation. Helper cell lineswhich have deletions of the packaging signal may be used. These celllines produce empty virions, since no genome is packaged. If aretroviral vector is introduced into such cells in which the packagingsignal is intact, but the structural genes are replaced by other genesof interest, the vector can be packaged and vector virion produced.

Other gene delivery mechanisms that can be used for delivery ofantisense sequences to target cells include colloidal dispersion andliposome-derived systems, artificial viral envelopes, and other systemsavailable to one of skill in the art. See, e.g., Rossi, 1995, Br MedBull 51(1):217-225; Morris et al., 1997, Nucleic Acids Res25(14):2730-2736; and Boado et al., 1998, J Pharm Sci 87(11):1308-1315.For example, delivery systems can make use of macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes.

In one embodiment, a method of the present invention administers atherapeutically effective amount of an antisense oligonucleotide havinga sequence capable of binding specifically with any sequences of an mRNAmolecule which encodes CTGF, so as to prevent translation of CTGF mRNA.

In another embodiment of the present invention, a method is provided inwhich a therapeutically effective amount of an antisense oligonucleotidehaving a sequence capable of binding specifically with any sequences ofCTGF mRNA so as to prevent translation of the mRNA.

Small Molecule Inhibitors. The present invention further provides amethod in which small molecules are used to inhibit the activity of CTGFby blocking the binding of responsive cytokines to the CTGF responsivereceptor. For example, the present invention provides methods oftreating and preventing kidney fibrosis utilizing small molecules thatmodulate, regulate and inhibit CTGF activity.

In order to identify small molecules and other agents useful in thepresent methods for treating or preventing a renal disorder bymodulating the activity and expression of CTGF, CTGF and biologicallyactive fragments thereof can be used for screening therapeutic compoundsin any of a variety of screening techniques. Fragments employed in suchscreening tests may be free in solution, affixed to a solid support,borne on a cell surface, or located intracellularly. The blocking orreduction of biological activity or the formation of binding complexesbetween CTGF and the agent being tested can be measured by methodsavailable in the art.

Other techniques for drug screening which provide for a high throughputscreening of compounds having suitable binding affinity to CTGF, or toanother target polypeptide useful in modulating, regulating, orinhibiting the expression and/or activity of CTGF, are known in the art.For example, microarrays carrying test compounds can be prepared, used,and analyzed using methods available in the art. See, e.g., Shalon, D.et al., 1995, International Publication No. WO95/35505, Baldeschweileret al., 1995, International Publication No. WO95/251116; Brennan et al.,1995, U.S. Pat. No. 5,474,796; Heller et al., 1997, U.S. Pat. No.5,605,662.

Identifying small molecules that modulate CTGF activity can also beconducted by various other screening techniques, which can also serve toidentify antibodies and other compounds that interact with CTGF and canbe used as drugs and therapeutics in the present methods. See, e.g.,Enna et al., eds., 1998, Current Protocols in Pharmacology, John Wiley &Sons, Inc., New York N.Y. Assays will typically provide for detectablesignals associated with the binding of the compound to a protein orcellular target. Binding can be detected by, for example, fluorophores,enzyme conjugates, and other detectable labels well known in the art.See, e.g., Enna et al., supra. The results may be qualitative orquantitative.

For screening the compounds for specific binding, various immunoassaysmay be employed for detecting, for example, human or primate antibodiesbound to the cells. Thus, one may use labeled anti-hIg, e.g., anti-hIgM,hIgG or combinations thereof to detect specifically bound human antibodyof the galactosyl epitope. Various labels can be used such asradioisotopes, enzymes, fluorescers, chemiluminescers, particles, etc.There are numerous commercially available kits providing labeledanti-hIg, which may be employed in accordance with the manufacturer'sprotocol.

For screening the compounds for cytotoxic effects, a wide variety ofprotocols may be employed to ensure that one has the desired activity.One will normally use cells, which may be naturally occurring ormodified, cell lines, or the like. The cells may be prokaryotic oreukaryotic. For example, if one is interested in a pathogen, where itdoes not matter to which epitope the compound conjugate binds, one cancombine the pathogenic cells with each of the compounds in the presenceof an antibody dependent cytotoxic system to determine the cytotoxiceffect. One may perform this assay either prior to or subsequent todetermining the effect of the various candidate compounds on cells ofthe host to whom the compound would be administered. In this way, onewould obtain a differential analysis between the affinity for thepathogenic target and the affinity for host cells which might beencountered, based on the mode of administration.

In some situations, one would be interested in a particular cellularstatus, such as an activated state, as may be present with T cells inautoimmune diseases, transplantation, and the like. In this situationone would first screen the compounds to determine those which bind tothe quiescent cell, and as to those compounds which are not binding tothe quiescent cells, and screen the remaining candidate compounds forcytotoxicity to the activated cells. One may then screen for other cellspresent in the host which might be encountered by the compounds todetermine their cytotoxic effect. Alternatively, one might employ cancercells and normal cells to determine whether any of the compounds havehigher affinity for the cancer cells, as compared to the normal cells.Again, one could screen the library of compounds for binding to normalcells and determine the effect. Those compounds which are not cytotoxicto normal cells could then be screened for their cytotoxic effect tocancer cells. Even where some cytotoxicity exists for normal cells, inthe case of cancer cells, where there is a sufficient differentiation incytotoxic activity, one might be willing to tolerate the lowercytotoxicity for normal cells, where the compound is otherwise shown tobe effective with cancer cells.

Instead of using cells which are obtained naturally, one may use cellswhich have been modified by recombinant techniques. Thus, one may employcells which can be grown in culture, which can be modified byupregulating or downregulating a particular gene. In this way, one wouldhave cells that differ as to a single protein on the surface. One couldthen differentially assay the library as to the effect of members of thelibrary on cells for which the particular protein is present or absent.In this way, one could determine whether the compound has specificaffinity for a particular surface membrane protein as distinct from anyof the proteins present on the surface membrane.

One may differentiate between cells by using antibodies binding to aparticular surface membrane protein, where the antibodies do notinitiate the complement dependent cytotoxic effect, for example, usingdifferent species, isotypes, or combinations thereof. By adding theantibodies, blocking antisera or monoclonal antibodies, to one portionof the cells, those cells will not have the target protein available forbinding to the library member. In this way one creates comparative cellswhich differ in their response based on the unavailability in one groupof a single protein. While antibodies will usually be the mostconvenient reagent to use, other specific binding entities may beemployed which provide the same function.

For use in the assay to determine binding, one may use anantibody-dependent cytotoxic system. One could use synthetic mixtures ofthe ingredients, where only those components necessary for the cytotoxiceffect are present. This may be desirable where components of blood orplasma may adversely affect the results of the assay.

Also, while a cellular lawn is an extremely convenient way to screenlarge numbers of candidates, other techniques may also find use. Thesetechniques include the use of multiwell plates, and the various devicesused for the preparation of the combinatorial library, such as pins, teabags, etc. One may grow the cells separately in relation to the natureof the various devices, where the device may then be contacted with thecells or have the cells grown on the device. The device may be immersedin an appropriate culture, seeded with the cells, or otherwise providedfor contact between the cells and the candidate compound. After addingthe cytotoxic agent, one may then analyze for lysis in a variety ofways. For example, FACS may be used for distinguishing between live anddead cells, [⁵¹Cr] release may be employed, or detection of anintracellular compound in the supernatant, may serve to detect activecompounds.

In addition, one may wish to know whether the compound has agonist orantagonist activity. The subject assay techniques provide for a rapidway for determining those compounds present in the library which bind tothe target protein. Once, one has substantially narrowed the number ofcandidate compounds, one can use more sophisticated assays for detectingthe activity of the compound itself. In this way, one can perform arapid screen to determine binding affinity and specificity, followed bya more intensive screen to determine activity. Various techniques existfor determining activity, where the cells may be modified, so that amarker gene will be activated which will provide for a detectablesignal. Conveniently, the signal may be associated with production of adye, the production of a surface membrane protein which can be detectedwith labeled antibodies, or the secretion of a protein which can bedetected in the supernatant by any of a variety of techniques. Forexample, the gene that is expressed may be luciferase modified to have aleader sequence so as to be secreted, whereby the supernatant can thenbe screened for light generation formation by using an appropriatesubstrate.

Various protocols may be employed for screening the library. To somedegree, this will depend upon the nature of the preparation of thecompounds. For example, the compounds may be bound to individualparticles, pins, membranes, or the like, where each of the compounds issegregatable. In addition, the amount of compound available will vary,depending upon the method employed for creating the library.Furthermore, depending upon the nature of the attachment of the compoundto the support, one may be able to release aliquots of a compound, so asto carry out a series of assays. In addition, the manner in which thecompounds are assayed will be affected by the ability to identify thecompound which is shown to have activity.

Where the compounds are individually on a surface in a grid, so that ateach site of the grid one knows what the composition is, one can providea cellular lawn which is similarly organized as a grid and may be placedin registry with the compounds bound to the solid surface. Once the lawnand solid substrate are in registry, one may release the compounds fromthe surface in accordance with the manner in which the compounds areattached. After sufficient time for the compounds to bind to theproteins on the cellular surface, one may wash the cellular lawn toremove non-specifically bound compounds. One or more washings may beinvolved, where the washings may provide for varying degrees ofstringency, depending upon the desired degree of affinity. After thewashings have been completed, mammalian blood or plasma may then beadded and incubated for sufficient time for cytotoxicity. The plasma orblood may then be removed and plaques observed, where the nature of thecompound can be determined by virtue of the position in the grid. Theplasma or blood can be free of any components that would naturally killthe cells of the lawn.

Since the preparative process may be repeated, one could prepare aplurality of solid substrates, where the same compounds are prepared atthe comparable sites, so that the screening could be repeated with thesame or different cells to determine the activity of the individualcompounds. In some instances, the identity of the compound can bedetermined by a nucleic acid tag, using the polymerase chain reactionfor amplification of the tag. See, e.g., International Publication No.WO93/20242. In this instance, the compounds that are active may bedetermined by taking the lysate and introducing the lysate into apolymerase chain reaction medium comprising primers specific for thenucleic acid tag. Upon expansion, one can sequence the nucleic acid tagor determine its sequence by other means, which will direct theselection of the procedure that is used to prepare the compound.

Alternatively, one may have tagged particles where the tags arereleasable from the particle and provide a binary code that describesthe synthetic procedure for the compounds bound to the particle. See,e.g., Ohlmeyer et al., 1993, Proc Natl Acad Sci USA 90:10922. These tagscan conveniently be a homologous series of alkylene compounds, which canbe detected by gas chromatography-electron capture. Depending upon thenature of the linking group, one may provide for partial release fromthe particles, so that the particles may be used 2 or 3 times beforeidentifying the particular compound.

While for the most part libraries have been discussed, any large groupof compounds can be screened analogously, so long as the CTGF epitopecan be joined to each of the compounds. Thus, compounds from differentsources, both natural and synthetic, including macrolides,oligopeptides, ribonucleic acids, dendrimers, etc., may also be screenedin an analogous manner.

Formation of a plaque in the assay demonstrates that binding of themember of the library to the cell, usually a surface protein, does notinterfere with the CTGF epitope binding to an antibody, that the immunecomplex is sufficiently stable to initiate the complement cascade, andthat the member has a high affinity for the target.

The subject methodology can be used in any situation where one has acellular target to be killed, particularly those cellular targets havinglow or no CTGF epitope. Thus, the cellular target may be a prokaryote,which is pathogenic. Various organisms include, for example,microbacterium, Yersinia, Pseudomonas, Bordetella pertussis, Treponemapallidum, Neisseria gonorrhoea, Streptococcus, Hemophilus influenza,etc. Other pathogens include eukaryotes, particularly fungi, such asCandida, Histoplasma, etc., and protozoa, e.g., Giardia. In addition,viruses which provide for surface membrane proteins in infected cells,can also be the target of the subject compounds, where the cells thatare screened have been vitally infected.

Host cells may also serve as targets, where the cells are eitherabnormal or act in an adverse way to the host or treatments of the host.For example, cancerous tissues which can be distinguished from normaltissue can serve as a target for the subject compounds. T or B cellsassociated with autoimmune diseases or associated with GVHD ortransplant rejection may also serve as targets. Aberrant cells,regardless of their nature, so long as they can be distinguished fromnormal cells, may also serve as targets. Thus, psoriatic lesions,lymphoma cells, bacterial, fungal, parasitic, virus infected cells, maybe targets of the subject products. Also, where one wishes to ablate aportion of cells, without removal of all of the cells, such as cellsexpressing a differentiation marker such as T cell subsets, activatedplatelets, endothelial cells, hormone or cytokine receptor expressingcells, the subject compounds may find application.

Other screening methods for obtaining small molecules that modulate theactivity of CTGF can be found, for example, International PublicationNo. WO 98/13353.

Compounds/Molecules. The present invention provides methods for treatingand preventing disorders associated with kidney fibrosis by modulating,regulating, or inhibiting the activity of CTGF. These methods cancomprise the administration of a therapeutically effective amount of acompound that blocks the binding interactions or blocks enzymes involvedin the signal transduction pathway of CTGF. More specifically, thepresent invention provides a method for inhibiting the activity of CTGFby administering compounds that block the induction of CTGF.

Compounds that modulate CTGF gene expression and/or CTGF activity in themethod of the invention include agents which cause an elevation incyclic nucleotides in the cell. Other compounds that may block theinduction of CTGF according to the methods of the present invention maybe identified using the screening methods described above.

In yet a further embodiment of the present invention, the methodprovides for the administration of molecules that interrupt thepost-translational modification of full length CTGF or block theactivation of an inactive precursor of CTGF. As discussed herein,exposure of mesangial cells to TGF-β resulted in the marked appearanceof additional bands at 28-30 kDa which correspond in size to thecarboxy- and amino-terminal halves of the full length CTGF molecule. Asdisclosed above, TGF-β treatment may result in the production ofproteases or other factors capable of cleaving the full-length molecule.Molecules that inhibit CTGF activity may be identified using thescreening methods provided herein.

The methods of the present invention may further be used to prevent ortreat fibrosis in the kidney associated with allograft rejectioncomprising administering a therapeutically effective amount of any oneof the agents described above.

The invention further provides a method for treating or preventingdiabetes by administering an effective amount of insulin and aneffective amount of an agent that regulates, modulates, or inhibits CTGFactivity as described above.

C. Pharmaceutical Formulations and Routes of Administration

Routes of Administration. The antibodies, small molecules, and othercompounds described herein can be administered to a human patient perse, or in pharmaceutical compositions comprising, where appropriate,suitable carriers or excipients. The present invention contemplatesmethods of treatment in which agents that modulate or regulate theexpression or activity of CTGF or fragments thereof are administered toa patient in need, in amounts suitable to treat or prevent theoverproduction of ECM associated with CTGF. The present methods oftreatment and prevention can comprise administration of an effectiveamount of the agent to a subject which is preferably a mammaliansubject, and most preferably a human subject. In a preferred embodiment,the subject mammal and the agent administered are of homologous origin.Most preferably, the subject and the agent administered are human inorigin.

An effective amount can readily be determined by routine experiment, ascan the most effective and convenient route of administration and themost appropriate formulation. Various formulations and drug deliverysystems are available in the art. See, e.g., Gennaro, ed., 1990,Remington 's Pharmaceutical Sciences, 18^(th) ed., Mack Publishing Co.,Easton Pa. Suitable routes of administration may, for example, includeoral, rectal, transmucosal, or intestinal administration and parenteraldelivery, including intramuscular, subcutaneous, intramedullaryinjections, as well as intrathecal, direct intraventricular,intravenous, intraperitoneal, intranasal, or intraocular injections. Thecomposition may be administered in a local rather than a systemicmanner. For example, a composition comprising an agent which modulates,regulates, or inhibits the activity of CTGF can be delivered viainjection or in a targeted drug delivery system into an area in whichthere is excess circulating CTGF or ECM overproduction or into an areain which inhibition of CTGF activity is desired, often in a depot orsustained release formulation.

The pharmaceutical compositions of the present invention may bemanufactured by any of the methods well-known in the art, such as byconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes. Asnoted above, the compositions of the present invention can include oneor more physiologically acceptable carriers such as excipients andauxiliaries which facilitate processing of active molecules intopreparations for pharmaceutical use. Proper formulation is dependentupon the route of administration chosen.

For injection, for example, the composition may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks's solution, Ringer's solution, or physiological saline buffer. Fortransmucosal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art. For oral administration, the compounds can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the compounds of the invention to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions and the like, for oral ingestion by a subject. The compoundsmay also be formulated in rectal compositions such as suppositories orretention enemas, e.g., containing conventional suppository bases suchas cocoa butter or other glycerides.

Pharmaceutical preparations for oral use can be obtained as solidexcipients, optionally grinding a resulting mixture, and processing themixture of granules, after adding suitable auxiliaries, if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinyl pyrrolidone (PVP). If desired,disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations for oral administration include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebuliser, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, orany other suitable gas. In the case of a pressurized aerosol, theappropriate dosage unit may be determined by providing a valve todeliver a metered amount. Capsules and cartridges of, for example,gelatin, for use in an inhaler or insufflator may be formulated. Thesetypically contain a powder mix of the compound and a suitable powderbase such as lactose or starch.

Compositions formulated for parenteral administration by injection,e.g., by bolus injection or continuous infusion can be presented in unitdosage form, e.g., in ampoules or in multi-dose containers, with anadded preservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Formulations for parenteral administration include aqueoussolutions of agents that affect the activity of CTGF or fragmentsthereof, in water-soluble form.

Suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil and synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Optionally, the suspension may also contain suitablestabilizers or agents that increase the solubility of the compounds toallow for the preparation of highly concentrated solutions.Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compositions of the present invention may also be formulated as adepot preparation. Such long acting formulations may be administered byimplantation (for example, subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the compounds may beformulated with suitable polymeric or hydrophobic materials (for exampleas an emulsion in an acceptable oil) or ion exchange resins, or assparingly soluble derivatives, for example, as a sparingly soluble salt.

Pharmaceutical carriers for the hydrophobic molecules of the inventioncould include co-solvent systems comprising, for example, benzylalcohol, a nonpolar surfactant, a water-miscible organic polymer, and anaqueous phase. The co-solvent system may be the VPD co-solvent system.VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the nonpolarsurfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made upto volume in absolute ethanol. The VPD co-solvent system (VPD:5W)consists of VPD diluted 1:1 with a 5% dextrose in water solution. Thisco-solvent system is effective in dissolving hydrophobic compounds andproduces low toxicity upon systemic administration. Naturally, theproportions of a co-solvent system may be varied considerably withoutdestroying its solubility and toxicity characteristics. Furthermore, theidentity of the co-solvent components may be varied. For example, otherlow-toxicity nonpolar surfactants may be used instead of polysorbate 80,the fraction size of polyethylene glycol may be varied, otherbiocompatible polymers may replace polyethylene glycol, e.g. polyvinylpyrrolidone, and other sugars or polysaccharides may substitute fordextrose.

Alternatively, other delivery systems for hydrophobic molecules may beemployed. Liposomes and emulsions are well known examples of deliveryvehicles or carriers for hydrophobic drugs. Certain organic solventssuch as dimethylsulfoxide also may be employed, although usually at thecost of greater toxicity. Additionally, the compounds may be deliveredusing sustained-release systems, such as semi-permeable matrices ofsolid hydrophobic polymers containing the effective amount of thecomposition to be administered. Various sustained-release materials areestablished and available to those of skill in the art.Sustained-release capsules may, depending on their chemical nature,release the compounds for a few weeks up to over 100 days. Depending onthe chemical nature and the biological stability of the therapeuticreagent, additional strategies for protein stabilization may beemployed.

Effective Dosage. For any composition used in the present methods oftreatment, a therapeutically effective dose can be estimated initiallyusing a variety of techniques well known in the art. For example, in acell culture assay, a dose can be formulated in animal models to achievea circulating concentration range that includes the IC₅₀ as determinedin cell culture. Where inhibition of CTGF activity is desired, forexample, the concentration of the test compound which achieves ahalf-maximal inhibition of CTGF activity can be determined. Dosageranges appropriate for human subjects can be determined using dataobtained from cell culture assays and other animal studies.

A therapeutically effective dose refers to that amount of the moleculethat results in amelioration of symptoms or a prolongation of survivalin a subject. Toxicity and therapeutic efficacy of such molecules can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., by determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio of toxic to therapeuticeffects is the therapeutic index, which can be expressed as the ratioLD₅₀/ED₅₀. Molecules which exhibit high therapeutic indices arepreferred.

Dosages preferably fall within a range of circulating concentrationsthat includes the ED₅₀ with little or no toxicity. Dosages may varywithin this range depending upon the dosage form employed and the routeof administration utilized. The exact formulation, route ofadministration, and dosage will be chosen in view of the specifics of asubject's condition.

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety which are sufficient to modulate orregulate CTGF activity as desired, i.e. minimal effective concentration(MEC). The MEC will vary for each compound but can be estimated from,for example, in vitro data, such as the concentration necessary toachieve 50-90% activity of CTGF to induce bone growth using the assaysdescribed herein.

Dosages necessary to achieve the MEC will depend on individualcharacteristics and route of administration. Compositions should beadministered using a regimen which maintains plasma levels above the MECfor about 10-90% of the duration of treatment, preferably about 30-90%of the duration of treatment, and most preferably between 50-90%. Incases of local administration or selective uptake, the effective localconcentration of the drug may not be related to plasma concentration.

The amount of composition administered will, of course, be dependent ona number of factors, including, but not limited to, the particularsubject's weight, the severity of the affliction, the manner ofadministration, and the judgment of the prescribing physician.

Packaging. The compositions may, if desired, be presented in a pack ordispenser device which may contain one or more unit dosage formscontaining the active ingredient. The pack may, for example, comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.Compositions comprising a compound of the invention formulated in acompatible pharmaceutical carrier may also be prepared, placed in anappropriate container, and labeled for treatment of an indicatedcondition. Suitable conditions indicated on the label may includetreatment of disorders or diseases in which cartilage or bone induction,wound healing, neuroprotection or the like is desired.

Receptor-Ligand Complexes. As a consequence of the above describedscreening techniques, as well as other known screening techniques whichmay be applied in the context of the present invention, CTGF-ligandcomplexes can be formed. These complexes may include complexes whereinthe ligand is a CTGF antagonist, a CTGF agonist, or any another compoundcapable of modulating the expression and activity of CTGF. Partialagonists or antagonists of the CTGF receptor may be useful fortherapeutic or diagnostic purposes. CTGF-agent complexes may be usefulas therapeutic entities in their own right or in methods of detectingand quantifying CTGF levels in a sample. The measurement andquantification of CTGF, for example, through the detection ofCTGF-ligand complexes, can be accomplished by methods available in theart. See, e.g., Enna et al., supra.

D. Diagnostics

The present invention is further directed to a method of detecting ordiagnosing the presence of pathology of a tissue characterized by anexcessive accumulation of the extracellular matrix components, inparticular, those associated with renal disorders. One method involvesthe detection or diagnosis of diabetes, including diabetic nephropathyand diabetic glomerulosclerosis. In a preferred method, the detection ordiagnosis is accomplished by measuring CTGF levels in a urine samplefrom a patient. In one embodiment, the method includes determining thelevel of CTGF in a first urine sample and comparing this level to thelevel of CTGF present in a normal urine sample, i.e., a sample from asubject without a renal disorder. An elevated level of CTGF in the firstsample is indicative of the pathological condition in question, forexample, diabetes or hypertension. In particular, individuals withoutany renal disorders, normal levels of CTGF may be at or close to zero.In diabetic patients or in patients experiencing infection or othertrauma, levels of CTGF may be significantly increased. Thus, thepresence of kidney fibrosis could be identified by detecting increasesin levels of CTGF in a sample. In a preferred method, the sample is anon-intrusive sample such as a urine sample. Assessment of CTGF levelsin a urine sample can be accomplished, for example, by ELISA using aCTGF-specific antibody. Detection of CTGF levels could be indicative ofthe advancement or worsening of diabetic hypertension or other renaldisorders prior to the onset of renal complications, providing for amethod of early-stage detection and diagnosis. Furthermore, CTGF levelscan serve as a predictor of, for example, which diabetic patients have apredisposition to develop kidney diseases and disorders.

More generally, detection of CTGF levels, including levels of uniqueforms or fragments of CTGF, may be obtained through immunoassay methods,for example, ELISAs, RIAs, or any other assays which utilize an antibodyto detect the presence of a protein marker. The ELISA and RIA methodsare preferred and may be used, for example, with the monoclonalantibodies of the present invention to detect levels of CTGF. In apreferred method of the invention, urine samples are obtained first frompatients suspected or known to have a renal disease or disorder. Levelsof CTGF in this first sample are measured, for example, throughimmunoassay, and are compared with the CTGF levels in a second sample,the second sample being obtained from a patient known to have a renaldisorder or from a patient known not to have any renal disorder, todetermine the presence or progression of a kidney disease. The samemethods may be used to monitor the progression of a kidney disease.

More generally, antibodies specific for a target polypeptide, such asantibodies specific for CTGF, are useful in the present invention fordiagnosis of renal disorders and diseases associated with aberrantexpression of CTGF. Diagnostic assays for CTGF can include methodsutilizing the antibody and a label to detect CTGF in a sample from apatient suspected of having a renal disorder or disease. The samplecould comprise, for example, body fluids, cells, tissues, or extracts ofsuch tissues, including, for example, glomeruli microdissected frombiopsy material. Protocols employed to screen for and identifyantibodies having the desired specificity can also be used for thedetection of CTGF or the target polypeptide in the sample.

Preferably, in the diagnostic methods of the present invention, normalor standard values for CTGF expression are established in order toprovide a basis for the diagnosis of the existence of a renal disease ordisorder or a predisposition to a renal disease or disorder. In one ofthe methods of the present invention, this is accomplished by combiningbody fluids or cell extracts taken from normal subjects with antibody toCTGF under conditions suitable for complex formation. Such conditionsare well known in the art. The amount of standard complex formation maybe quantified by comparing levels of antibody-target complex in thenormal sample with a dilution series of positive controls, in which aknown amount of antibody is combined with known concentrations ofpurified CTGF. Standard values obtained from normal samples may becompared, for example, in a specific embodiment, with values obtainedfrom samples from subjects suspected of having a kidney disease ordisorder, or having a predisposition to a kidney disease or disorder,associated with kidney fibrosis. Deviation between standard and subjectvalues establishes the presence of or predisposition to the diseasestate. The diagnostic methods of the present invention may also bedirected to the detection of a predisposition or susceptibility to arenal disorder. This can be accomplished, for example, by detecting amarker indicative of a predisposition or susceptibility to develop aparticular disorder, for example, diabetes. The marker can comprise, forexample, a genetic polymorphism.

Monoclonal antibodies can be detected by methods discussed, for example,supra. Monoclonal antibodies against CTGF can be conjugated to anappropriate enzyme such as horseradish peroxidase, protein ferritin,enzyme alkaline phosphatase, β-D-galactosidase etc. These enzyme-linkedantibody preparations can be mixed with, for example, urine samples thatcontain unknown amounts of CTGF in an indirect ELISA. Direct or sandwichELISAs could also be performed using the same antibodies.

RIA techniques may also be used to measure levels of CTGF in, forexample, urine. For example, CTGF may be radioactively labeled and mixedwith monoclonal antibodies specific for CTGF and a serum samplecontaining an unknown amount of unlabeled CTGF. Binding competitionbetween the labeled and unlabeled CTGF with the monoclonal antibodyoccurs. By measuring the amount of radioactivity of the reactionmixture, the amount of CTGF present in the sample can be quantitativelydetermined. See, e.g., U.S. Pat. Nos. 4,438,209 and 4,591,573.Non-competitive RIAs can also be performed.

Polynucleotide sequences encoding CTGF can be used for the diagnosis ofconditions or diseases associated with increased levels of CTGFexpression. For example, polynucleotide sequences encoding CTGF may beused in hybridization or PCR assays of fluids or tissues from biopsiesto detect CTGF expression. The form of such qualitative or quantitativemethods may include Southern or northern analysis, dot blot or othermembrane-based technologies; PCR technologies; dip stick, pin, chip andELISA technologies. All of these techniques are well known in the artand are the basis of many commercially available diagnostic kits.

The present invention additionally provides methods for evaluating theeffectiveness of anti-fibrotic therapy, including the use of ACEinhibitors, by measuring the levels of CTGF in a sample from a subjectundergoing a course of treatment for diseases and disorders associatedwith fibrosis. CTGF levels can be measured in samples, for example,urine sample, taken from the subject at various points before, during,and after a course of treatment. The efficacy of a treatment can beevaluated with reference to the variation in CTGF levels present in thesamples taken at different stages of a course of treatment.

Kits. The present invention provides kits for detecting CTGF in samples,in particular, in fluid samples. In a preferred embodiment, thediagnostic kits of the present invention contain reagents for measuringlevels of CTGF in urine samples. In a particular embodiment, this kitcomprises a monoclonal antibody specific for CTGF bound to a support anda second monoclonal antibody specific for a different CTGF epitope andenzyme-labeled. The kit further comprises reagents for detecting theenzyme-labeled monoclonal antibody. The reagent kit employsimmunological methods in measuring CTGF in the urine sample, thusallowing for the detection and monitoring of kidney disorders anddiseases. In particular embodiments, the kit allows for the detectionand monitoring of fibrotic and sclerotic disorders resulting from, forexample, diabetes and hypertension. In another embodiment, the kitcomprises a radio-labeled or fluorescein-labeled antibody in place ofthe enzyme-labeled antibody.

In one embodiment, the diagnostic kit of the present invention compriseselements useful in the detection of CTGF in tissue samples, usingimmunohistochemical techniques. The kit could be used in conjunctionwith, for example, a software program which allows for quantitativemeasurement of the levels of CTGF in the tissue sample by image analysisor other comparative techniques. See, e.g., Riser et al., 1996, supra.Another embodiment provides a diagnostic kit for detecting and measuringlevels of CTGF mRNA in tissue samples. In one embodiment, the kitcomprises reagents used to reverse transcribe CTGF mRNA to DNA. The kitcan further comprise reagents necessary to amplify CTGF-specific DNA,including primers complementary to polynucleotides encoding CTGF orfragments thereof. The kit can also include a competitive mimic ormutant cDNA for use in quantifying the level of CTGF mRNA present in thesample.

In a preferred embodiment, the diagnostic kit of the present inventionis packaged and labeled, for example, in box or container which includesthe necessary elements of the kit, and includes directions andinstructions on the use of the diagnostic kit.

The following examples explain the invention in more detail. Thefollowing preparations and examples are given to enable those skilled inthe art to more clearly understand and to practice the presentinvention. The present invention, however, is not limited in scope bythe exemplified embodiments, which are intended as illustrations ofsingle aspects of the invention only, and methods which are functionallyequivalent are within the scope of the invention. Indeed, variousmodifications of the invention in addition to those described hereinwill become apparent to those skilled in the art from the foregoingdescription and accompanying drawings. Such modifications are intendedto fall within the scope of the appended claims.

EXAMPLES

Unless otherwise stated, the following materials and methods were usedin the examples of the present invention.

Cells And Tissue Culture. The mesangial cells were a cloned-line derivedfrom outgrowths of Fischer rat glomeruli, and upon serial passage, thesemesangial cells continue to express key markers. (See, e.g., Riser etal., 1998, J Am Soc Nephrol 9:827-836.) The medium used was RPMI 1640with penicillin and streptomycin and, unless otherwise noted, 5 mMglucose. The growth medium contained 20% NU-SERUM media supplement(Collaborative Research, Bedford Mass.). Unless otherwise noted,mesangial cells were cultured for approximately 4 days in growth medium,and when reaching confluency, were washed twice with serum-free mediumand incubated for 2448 hours under serum-deprived of 0.5% FCS (fetalcalf serum) conditions. The cultures were then incubated for adesignated period in fresh maintenance medium (0.5% FCS), with orwithout experimental treatments. At the concentration of FCS used inthese studies, no active TGF-β1, TGF-β2, or TGF-β3 was detectable in thefresh medium, as determined by a highly sensitive mink lung bioassay.(See, e.g., Riser et al., 1996, supra.) The renal fibroblasts used inNorthern analysis were mouse tubulointerstitial fibroblasts (TFB). (See,e.g., Alverez, et al., 1998, Kidney Int 41:14-23.)

Animals And Specimen Collection. Diabetic male db/db mice and theirnondiabetic db/m littermates were obtained from Jackson Laboratories(Bar Harbor Me.). The db/db mouse carries a defective receptor gene forleptin, a key weight control hormone. (See, e.g., Hummel et al., 1966,Science 153:1127-1128.) These mice become obese at 3 to 4 weeks of ageand develop hyperglycemia. Associated nephropathy includes proteinuriaand mesangial expansion with increased mesangial matrix that develops by5 to 7 months. (See, e.g., Cohen et al., 1995, J Clin Invest95:2338-2345.) In the present experiments, mice were sacrificed at theage of 5 months. Blood glucose levels were determined during the studyand at sacrifice, using a calorimetric method based on the glucoseoxidase-peroxidase reaction and supplied in a kit form (GlucoseProcedure No. 510 kit, Sigma Diagnostics, St. Louis Mo.). Following a 24hour acclimation to metabolic cages, two consecutive 24 hour urinesamples were collected. At the end of the collection period, the lowerpart of the cage including the collection funnel was rinsed withdistilled water and the final sample volume recorded. Proteinconcentration in the urine was measured according to a method forquantifying microgram quantities of protein utilizing protein-dyebinding. (See, e.g., Bradford, 1976, Anal Biochem 72:248-254.)

After anesthesia by an oxygen/ether mixture, the abdominal cavity wasopened, a 23 gauge needle was inserted into the aorta and the kidneyswere perfused with four (4) ml of ice cold perfusion buffer of (RPMIwith 4% BSA) containing 10 mM vanadyl ribonucleoside complex (VRC), anRNase inhibitor (Gibco/BRL, Grand Island, N.Y.). Chilled 0.9% saline waspoured over the kidneys during this perfusion. The kidneys were thenremoved, and the right kidney was frozen in liquid nitrogen forsubsequent RNA extraction and Northern analysis. Fine sagittal slices ofthe left kidney were rapidly obtained. One section was fixed in 3.8%paraformaldehyde, embedded in parafilm and stained with periodic acidSchiff (PAS) for light microscopic evaluation. The remaining slices wereused for glomerular microdissection and reverse transcription andpolymerase chain reaction (RT-PCR) of the isolated glomeruli. Themethods used were a modification of known methods for determiningglomerular mRNA levels. (See, e.g., Peten et al., 1993, Kidney Int Suppl39:S55-S58.) Tissue sections were placed in a buffer of HBSS containing10 mM VRC, and then 50 glomeruli were dissected from each kidney in lessthan 50 minutes. The glomeruli were next transferred to a PCR tube with30 μl of rinse buffer (HBSS containing 5 mM DTT and 50 units/ml of humanplacental ribonuclease inhibitor (Boeringer Mannheim, IndianapolisInd.). Following centrifugation, the supernatant was removed andmicroscopically examined for the accidental presence of glomeruli. Sevenmicroliters of a lysis solution (rinse buffer containing 2% TritonX-100) were added, and the samples were stored at −70° C. untilprocessed. All of these procedures were carried out at 4° C.

Experimental samples from control and diabetic mice were thawed on iceand then subjected to 2 additional freeze/thaw cycles to lyse theglomeruli. The RT reaction was then carried out using a cDNA synthesiskit (Boehringer Mannheim), with oligo(dT) as a primer. Reactionscontaining glomeruli, but without added reverse transcriptase, orwithout glomeruli, but with reverse transcriptase, served as negativecontrols. The reaction mixture was incubated for 60 minutes at 42° C.,and then chilled to 4° C. for 10 minutes. Samples were then diluted at aratio of 1:10 in distilled water and frozen at −70° C. until PCR wascompleted.

Evaluation Of Renal Tissue By Light Microscopy. Five to 6 nonconsecutive6 μm sections per kidney were PAS stained and examined. Mesangialsclerosis was scored on a scale of zero to four (0-4), wherein zero (0)represents no lesion; one (1) represents minimal mesangial expansion;two (2) represents mesangial expansion and/or basement membranethickening; three (3) represents marked mesangial thickening, somecollapsed lumina, and occasional lobule with full sclerosis; and four(4) represents a diffuse collapse of capillary lumina, and sclerosisinvolving 75% or more of the tuft. A total of 100-150 glomeruli perkidney were scored by an observer blinded as to the origin of thespecimens. Only glomerular profiles showing a mesangial region thatcould be unequivocally evaluated were scored.

Competitive PCR and Northern blotting. All PCR were performed using theGENEAMP DNA amplification kit (Perkin-Elmer Cetus, Norwalk Conn.) and a9600 thermal cycler (Perkin Elmer). For quantitation, a competitive PCRreaction was run using a cDNA mimic. Thirty-eight cycles of replicationwere used. Five PCR tubes were set up for each sample. Each tube in aseries contained a fixed amount of the wild-type cDNA along withdecreasing concentrations of the mimic cDNA. The products were separatedby agarose gel electrophoresis and visualized by ethidium bromidestaining. Bands were digitized by scanning densitometry (SCANMASTER 3+densitometer; Howtek, Hudson N.H.) and quantified with image analysis(NIH Image, v. 1.59 from Twilight Clone BBS, Silver Springs Md.). A plotof the ratio of wild type/mimic vs. the reciprocal of the input mutantconcentration was constructed and the amount of glomerular cDNAdetermined from the resulting linear regression. Northern analysis wascarried out as previously described, following pulverization of samplesin a liquid-nitrogen cooled stainless-steel mortar and homogenization in1.0 ml of RNA STAT-60 reagent (Tel-Test Inc., Friendswood Tex.). Probesfor individual mRNAs and the corresponding cDNAs, were labeled with ³²Pby random hexamer priming using the PRIME-1 kit (Sigma). Autoradiogramswere digitized by scanning densitometry and quantified as describedabove.

Primers, probes, and cDNA mimics. Primers for CTGF were designed andsynthesized based on conserved sequences between the human and mouseCTGF (fisp 12) gene. The primers, Primer R and Primer F, were asfollows: Primer F: 5′-GAG TGG GTG TGT GAC GAG CCC AA G G-3′ and PrimerR: 5′ ATG TCT CCG TAC ATC TTC CTG TAG T-3′. The amplification productwas 558 bp in size. The sequence was confirmed by cloning into a PCRscript (Invitrogen Corp., Carlsbad Calif.). Two clones were sequencedand were identical. A competitive cDNA mimic was produced using a PCRmimic construction kit (K 1700-1, Clontech Laboratories, Palo AltoCalif.). For each mimic, two composite primers (3′ and 5′) were firstmade containing the CTGF target gene sequence, plus a 20-nucleotidestretch designed to hybridize to opposite strands of a heterologous DNAfragment provided in the kit. The desired primer sequences were thenincorporated into this fragment during PCR amplification. A dilution ofthe first PCR reaction was then amplified using only the gene-specificprimers. This ensured that all mimic molecules had completegene-specific sequences. The mimic was then purified by passage throughCHROMA SPIN TE-100 columns (Clontech). By this method, the size (200-650bp) could be adjusted by choosing the appropriate sequences of thegeneric DNA fragment for the composite primers. The resulting cDNAcompetes on an equal basis for the same primers in the same reaction. Anamplimer of the CTGF mimic was 496 bp in size.

Primers (Primer F and Primer R) for rat fibronectin cDNA were Primer F:5′ TGC CAC TGT TCT CCT ACG TG 3′ and Primer R: 5′-ATG CTT TGA CCC TTACAC GG 3′. A competitive mimic for fibronectin was constructed asdescribed above. Products of amplification were approximately 312 bps(sample) and 474 bps (mimic). Primers for GAPDH (Clontech) produced anamplification fragment of 985 bp. A GAPDH competitive mimic wasconstructed as described above, and produced a fragment of 604 bp. ThecDNA probe for Northern analysis was from a sequence of human CTGFshared by rat and mouse.

Production of recombinant CTGF and anti-CTGF antibodies. Recombinanthuman CTGF protein (rhCTGF) was generated using a baculoviral expressionsystem. A human CTGF open reading frame of 1047 bp was amplified usingprimers engineered with BamHI sites immediately flanking the ATG startcodon and TGA stop codon (forward primer 5′-GCT CCG CCC GCA GTG GGA TCCATG ACC GCC GCC-3′; reverse primer 5′-GGA TCC GGA TCC TCA TGC CAT GTCTCC GTA-3′). A clone designated as Clone DB60R32 was used as a template,which contains the entire 2075 bp CTGF cDNA. The amplified product wassubcloned into the BamHI site of PFASTBAC1 vector (Gibco/BRL), analyzedfor insert orientation, and verified by sequencing of both DNA strands.Generation of recombinant baculovirus containing the CTGF cDNA wasperformed as outlined by Gibco/BRL (pFastBac expression system).Recombinant baculovirus stocks were isolated, expanded to high virustiter, and used to infect High Five insect cells for expression of CTGF.The recombinant CTGF was purified by heparin sepharose affinitychromatography as described previously. Peak fractions containing rhCTGFwere determined by immunoblotting and Coomassie staining of sodiumdodecyl sulfate (SDS)-polyacrylamide gels.

Two anti-CTGF antibodies were used. The first, anti-CTGF polyclonaldesignated, pAb839, was prepared by immunizing rabbits with a keyholelimpet hemocyanin-coupled synthetic peptide corresponding to amino acids329-343 (CPG DND IFE SLY YRK) that is unique to the carboxy terminus ofCTGF. The production of antibody was monitored by ELISA with the peptideconjugated to BSA and absorbed to plastic. The anti-CTGF antibodies wereaffinity-purified by passage through a CPG DND IFE SLY YRK-Sepharosepeptide column using standard protocols (See, e.g., Harlow and Lane,1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor N.Y.). Peptide blocking studies confirmed themonospecificity of pAb839 for CTGF in western immunoblotting assays.Western immunoblot analysis also revealed that pAb839 recognized CTGFonly in a reduced conformation. The second antibody, pIgY3 polyclonal,was raised in chickens by immunizing with purified baculovirus-derivedfull-length rhCTGF protein, and was subsequently affinity purifiedthrough a rhCTGF-Sepharose column. (See, e.g., Kothapalli et al., 1997,Cell Growth Differ 8:61-68).

ELISA. The amount of specific extracellular matrix components secretedinto the culture medium was quantified by ELISA, using proceduresdescribed in the art. (See, e.g., Riser et al., 1992, J Clin Invest90:1932-1943.) It was previously determined in mesangial cell cultures,that media containing 0.5-1% FCS was optimal for the recovery offibronectin and collagen. (See, e.g., Riser et al., 1992, supra.)Experimental samples of culture medium were tested in triplicate.Purified matrix components, diluted in the same medium, were run(0.5-500 ng/well) as standards. All antisera were tested for specificitybefore their use by immunoblotting, with and without blocking, using theextracellular matrix standards. Color intensity was measured with aTITERTEK MULTISCAN MCC/340 plate reader (Flow Laboratories, McLean Va.,and the results analyzed using a curve-fitter computer program(Interactive Microware Inc., State College Pa.).

An indirect ELISA was used to quantitate CTGF levels in the conditionedmedia. Microtiter wells were coated with media samples or the rhCTGFstandard (50 μl/well) for 2 hours at room temperature in a 96-wellplate. The wells were washed 4 times with Dulbecco's phosphate bufferedsaline (D-PBS) and then incubated with pIgY3 antibody at 1.25 μg/ml (50μl/well) in a blocking buffer of 1% BSA, 0.05% Tween 20 in D-PBS for 60minutes. After thorough washing with D-PBS, an HRP-conjugated rabbitanti-chicken IgG (Zymed Laboratories Inc., South San Francisco Calif.)was added to all wells at a 1:6400 dilution in blocking buffer for 30minutes. The substrate, TMB-ELISA (Gibco/BRL) was added at roomtemperature for 15 minutes. The reaction was stopped with 1 M sulfuricacid and the color developed measured at 450 nm in an ELISA multiscanspectrophotometer (Molecular Devices, Sunnyvale Calif.). The amount ofCTGF protein present in samples was determined by using a logarithmicstandard curve using serial dilutions of 3 pg to 3 ng/well of rhCTGFstandard antigen.

Heparin sepharose precipitation and immunoblotting. To analyze for CTGFprotein expression, conditioned media were collected and theheparin-binding proteins precipitated by end-over-end mixing for 4 hoursat 4° C. with Heparin Sepharose CL-6B beads (Pharmacia, Pisctaway N.J.).The beads were washed three times with an ice cold RIPA lysis buffercomprised of 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1% Triton X-100, 1%deoxycholate, 0.1% SDS and 2 mM EDTA. The bound proteins were theneluted by boiling in a SDS sample buffer comprised of 62 mM Tris-HCl, pH6.8, 2.3% SDS, 10% glycerol and bromophenol blue for 5 minutes undereither non-reducing, or reducing conditions containing 5%mercaptoethanol. The eluted heparin-binding proteins were resolved in4-20% SDS-polyacrylamide gels and electrophoretically transferred tonitrocellulose filters (Schleicher and Schuell, Keene N.H.) for 2 hoursat 140 mA. The filters were blocked with blocking buffer comprised ofTTBS; 150 mM NaCl, 50 mM Tris, 0.2% Tween-20, 5% BSA, pH 7.4 for 2 hoursat room temperature, and then probed for CTGF by incubation for 40minutes with an anti-CTGF antibody at 0.5 μg/ml in the blocking buffer.After extensive washing at 37° C., the filters were incubated witheither a HRP-conjugated donkey anti-rabbit IgG (Amersham, ArlingtonHeights Ill.), or a HRP-conjugated rabbit anti-chicken IgG (Zymed) at a1:12,000 dilution in the blocking buffer. Immunoreactivity was detectedby using a SUPERSIGNAL chemiluminescent substrate (Pierce, RockfordIll.).

Additional reagents. Purified extracellular components used as standardswere rat collagen I (Upstate Biotechnology Inc., Lake Placid N.Y.) andrat fibronectin (Chemicon International Inc., Temecula Calif.). Thecorresponding antibodies, polyclonal anti-rat collagen I and anti-ratfibronectin were used in ELISA. In preliminary experiments, thepolyclonal anti-rat collagen I antibody did not cross-react withfibronectin or laminin, whereas the anti-rat fibronectin antibody didnot cross-react with collagen I or laminin. The TGF-β used forstimulation experiments was human TGF-β2 (Celtrix Corporation, SantaClara Calif.). This recombinant cytokine was produced in Chinese hamsterovary (CHO) cells and then purified by previously reported techniques(See, e.g., 1991, Ogawa et al., 1991, Meth Enzymol 198:317-327). Amonoclonal antibody designated as I.D 11.1, neutralizes TGF-β1, TGF-β2and TGF-β3 (Genzyme Corporation, Cambridge Mass.).

Statistical analysis. Data was expressed as means±SEM (Standard ErrorMean). For tissue culture data, unless otherwise noted, differencesbetween two groups were evaluated using a paired Student's t-test. Apaired test was utilized because of the cloned nature of the mesangialcells studied. In the case where results were normalized tocorresponding control values, the data was analyzed by a one-sample ttest with a hypothesized mean of 100% to compare the test group with thecontrol. A paired two-sample t test was used to examine differencesbetween 3 test groups. In both cases, a Holm's test was then appliedpost hoc to adjust for multiple comparisons. (See, e.g., Holm, 1979,Scan J Statist 6:65-70). For histological data, the mean sclerosis scorewas calculated in the glomeruli of each kidney and the statisticaldifference between the diabetic and control groups determined in anon-paired t-test.

A. CTGF is an Important Factor in the Pathogenesis of Renal Diseases,Including Diabetes

The examples provided in the present invention, provides the firstevidence demonstrating the production of CTGF protein by glomerularcells and its role as a potentially important factor in the pathogenesisof diabetic glomerulosclerosis. Hyperglycemia and glomerularhypertension are two major, well-known, casual factors of diabeticglomerulosclerosis. Prior to the present invention, the role of CTGF inthe development of glomerulosclerosis has not been studied. Thefollowing examples, demonstrate that CTGF stimulates cultured mesangialcells to produce, deposit, and accumulate extracellular matrixcomponents. The examples also demonstrate that the induction ofendogenous CTGF is triggered by increased glucose concentrations,exogenous TGF-β, and mechanical strain.

As demonstrated in Example 2, CTGF mRNA is expressed in the whole kidneyof normal animals, and that its level is high in comparison to the heartand brain, suggesting that endogenously produced CTGF may be involved inthe normal turnover of renal extracellular matrix. However, the lowlevels of constitutive CTGF mRNA expression demonstrated in culturedmesangial cells suggest that this cell type may have a controllingmechanism for CTGF formation different from that in the cells formingthe bulk of the renal tissue, i.e. tubular epithelial cells. The lowexpression of CTGF mRNA observed in mesangial cells under unstimulatedconditions is associated with an apparent release of small quantities ofCTGF protein into the culture medium, see Example 3. The CTGF proteinwas present as a 36 and 38 kD molecular species. The larger protein isequivalent in size to the full-length CTGF molecule predicted from geneanalysis, whereas the smaller peptide may represent a differentialN-glycosylation in the CTGF N-terminal half. It was observed that inboth insect and mammalian cells, pretreatment with tunicamycin, thatinhibits the N-glycosylation of glycoproteins, the larger CTGF band isreduced in its migration to localize with the smaller moiety. Thesemolecular species observed in mesangial cells are similar in size tothat secreted by vascular endothelial and fibroblast cells (See, e.g.,Bradham et al., 1991, supra; Kothapalli et al., 1997, supra; and Steffenet al., 1998, Growth Factors 15:199-213.) The small amounts of CTGFdetected in the conditioned medium of mesangial cultures were not theresult of low levels of its synthesis, but were rather due to therestricted release of the protein into medium. This was indicated by theability of sodium-heparin to dramatically increase the levels of CTGFprotein measured in the media. (See Example 3.) These results suggestthat as much as 80% of the CTGF synthesized by mesangial cells remainscell- or matrix-bound. In a quantitative assay it is shown that, in thepresence heparin, mesangial cells secrete approximately 7 ng of CTGF per10⁶ cells in each 24 hour period. (See Example 3.)

Given that CTGF stimulates extracellular matrix accumulation, it wasexamined whether known factors implicated in the development of diabeticglomerulopathy alter CTGF mRNA expression. High extracellular glucoseconcentrations markedly increased the levels of CTGF mRNA as well as theproduction of CTGF protein in mesangial cells. (See, Example 4.) In asimilar manner, TGF-β also upregulates the expression of CTGF mRNA andprotein. With strong upregulation, as occurred in response to TGF-β,there was a marked induction of a small molecular weight CTGF species,which according to its size of ˜18 kD, is approximately half of thefull-length CTGF molecule. (See, Example 2 and FIG. 6.) The size andproperties of this small molecular weight CTGF species recovered from aheparin-sepharose column indicated that it contains both thethrombospondin 1 and the C-terminal modules of CTGF. The small molecularspecies demonstrated in mesangial cells following stimulation, may havedistinct biological activities as compared to the whole molecule. SinceTGF-β secretion in mesangial cells is stimulated by increased ambientglucose concentrations, the observed induction of CTGF by high glucosemay occur indirectly, mediated by the action of TGF-β. Theneutralization studies described in Example 5 demonstrated a direct rolefor the cytokine in the process, since incubation with TGF-β antibodiesresulted in a complete blockade of CTGF stimulation. (See FIG. 8.)

Cyclic mechanical strain was also examined as a possible regulatoryelement in CTGF expression. The results demonstrated that stretching isa potent stimulus for the upregulation of CTGF mRNA levels (See Example7 and FIG. 10). The rapid induction of CTGF mRNA following stretchsuggests that TGF-β production and/or activity may not be required tomediate the initial effects of mechanical strain. Cyclic strain inducesTGF-β1 synthesis and activation, but this effect is only evident after48-72 hours of mechanical stimulation. (See, e.g., Riser et al., 1992,supra). These studies also demonstrated that TGF-β and CTGF are able toautoinduce their own expression in mesangial cells. (See, Example 3 andFIG. 3.) This autoinducing action of CTGF is the first time that suchaction has been observed for CTGF. Furthermore, this action appears tobe selective, since exogenous CTGF has no effect on TGF-β transcriptlevels. (See, FIG. 3.) These findings suggested that once stimulated byTGF-β, CTGF mRNA levels in mesangial cells may remain elevated even inthe absence of additional TGF-β activity resulting in a continuedenhancement of extracellular matrix synthesis and deposition, which mayexplain the prevalent inability to totally block extracellular matrixproduction in mesangial cells and in the mesangium by TGF-βneutralization. (See, e.g., Border et al., 1990, supra; Sharma et al.,1996, supra; and Ziyadeh et al., 1994, supra.)

Quantitative glomerular expression of CTGF mRNA in db/db mice (See,Example 10), demonstrated that CTGF action is a factor in the initiationof glomerular extracellular matrix deposition in diabetes. While CTGFmRNA is expressed in normal glomeruli, the levels are dramaticallyupregulated by 28-fold, after a short period of diabetes and before theonset of overt glomerular disease (See Example 10). As demonstrated inthese examples, CTGF mRNA upregulation occurred at a time whenglomerular fibronectin mRNA levels were increased. However, theglomerular mesangial expansion was minimal and proteinuriainsignificant. As compared to glomeruli, the much lower upregulation ofCTGF observed in the whole kidney as demonstrated in Example 10 showsthat the CTGF is, at least in the early phases of nephropathy, primarilyinvolved in the induction of the glomerular alterations. However, in themore advanced stages of diabetic nephropathy, CTGF may be an importantinducer of tubulointerstitial disease.

In summary, the following examples essentially demonstrate that, inaddition to enhanced glomerular TGF-β expression, CTGF upregulation isan important factor in the excess deposition of the extracellular matrixby mesangial cells. This CTGF upregulation is driven by a combination ofhigh glucose concentrations and cellular mechanical stain via pathwaysthat are both dependent and independent of TGF-β stimulation.

B. Experimental Data Demonstrating Nexus Between the Presence of CTGFand the Onset and Progression of Renal Disorders, Including Diabetes

Example 1

CTGF-Induced Changes In Extracellular Matrix Production Of MesangialCells. To determine the effects of exogenous CTGF on mesangial cellproduction of the extracellular matrix, serum-depleted cells wereexposed for 48 hours to media containing 20 ng/ml of rhCTGF. Forcomparison purposes, additional cultures were incubated in media withoutexogenous CTGF, but containing either 2 ng/ml of TGF-β, or 20 mMglucose. As anticipated, exogenous TGF-β and the high glucoseconcentration increased the amount of secreted fibronectin by 23 and30%, respectively, over that of controls as shown in FIG. 1A. Thepresence of exogenous CTGF in the media also effectively stimulatedfibronectin secretion by 45%. Like fibronectin, the quantity of secretedcollagen type I was also increased by 64% CTGF, as well as by 50% TGF-βor 22% high glucose as shown in FIG. 1B.

Example 2

Renal And Mesangial Cell CTGF Expression: Regulation By TGF-β. It wasdetermined whether cultured rat mesangial cells expressed CTGF mRNA, andthe results were compared to those from whole kidney. Northern analysisdemonstrated a single 2.4 kb CTGF transcript in mesangial cells andwhole kidney, but in contrast no detectable message was evident incultured kidney fibroblasts as demonstrated in FIG. 2. When compared toother tissues, the most abundant expression was in the kidney, beingapproximately 20-fold higher than in the brain.

To determine if TGF-β was a regulatory factor in mesangial cellexpression of CTGF message, cells were serum-depleted, exposed to 2ng/ml of TGF-β for 24 hours, the mRNA was then probed. Changes in TGF-βtranscript levels were also monitored. Exogenous TGF-β exposureincreased the expression of CTGF mRNA greater than 4-fold as shown inFIG. 3A and FIG. 3B, whereas TGF-β mRNA increased 80% (see FIG. 3A andFIG. 3C). To determine whether CTGF was capable of regulating its ownexpression, or that of TGF-β, mesangial cells were also exposed to 20ng/ml of rhCTGF. As shown in FIG. 3A and FIG. 3C, this treatment did notalter the level of TGF-β mRNA, but in contrast, strongly autoinducedCTGF message as demonstrated in FIG. 3A and FIG. 3B.

To demonstrate whether low CTGF mRNA expression in unstimulatedmesangial cells was associated with a detectable production of thecorresponding protein, and to determine the effects of TGF-β, cells wereserum depleted and then cultured for an additional 24 hours in freshmaintenance medium in the presence or absence of 2 ng/ml exogenousTGF-β. The conditioned medium was subsequently heparin-sulfateprecipitated and analyzed by immunoblotting using two differentanti-CTGF antibodies. Immunoblotting with pIgY3 antibody, raised againstthe full-length rhCTGF, demonstrated that under basal, unstimulatedconditions mesangial cells secreted very small amounts of CTGF (see FIG.4A). However, upon exposure to TGF-β, the secretion of CTGF protein wasmarkedly stimulated. The predominant product detected in these culturesmigrated to the same position as the recombinant standard.Immunoblotting of the same samples with the pAb839 antibody, raisedagainst a 15 amino acid sequence unique to CTGF, confirmed the identityof the protein detected (see FIG. 4B).

Example 3

Detection Of CTGF In Mesangial Cells. The CTGF protein detected Inmesangial cell cultures above represents free molecules present in themedia. The existence of a heparin binding domain within CTGF suggeststhat a substantial portion of the synthesized and released proteinexists bound to proteoglycans, or to fibronectin, present on the cellsurface or in the extracellular matrix. To ascertain whether this wasthe case, and to determine the time course for appearance of CTGF in theextracellular environment under unstimulated conditions, mesangial cellcultures were serum-deprived and then fresh maintenance media containing50 μg/ml of sodium heparin was added. Conditioned media were collectedafter defined incubation periods and the majority of the sample pooledand heparin-sulfate precipitated. Immunoblotting of the 4 hour samplesproduced faint CTGF bands at approximately 36 and 39 kD (see, FIG. 5A).The intensities of these bands increased sharply by 24 hours andremained elevated throughout the 72 hour incubation period. At 48 and 72hours, when the full-length CTGF bands were intense, a faint additionalband with an electrophoretic mobility of approximately 20 kD could alsobe detected. As previously demonstrated, in the absence of sodiumheparin, the CTGF present in the media was barely detectable, suggestingthat the majority of CTGF protein produced was bound to the cell and/orsubstrate. Because immunoblotting is largely a qualitative assay,individual supernatants were also evaluated by ELISA prior to theirpooling and precipitation, and the results were expressed on a per cellbasis. This highly quantitative assay demonstrated a time dependentincrease in CTGF, with approximately 7 ng/10⁶ cells being secreted in a24-hour period as shown in FIG. 5B. The amount of CTGF secreted in themedium during the total 72-hour period was reduced to 20% in the absenceof heparin.

In a subsequent experiment, the regulation of secreted CTGF by TGF-β wasreexamined, in the presence of heparin. Accordingly, mesangial cellswere serum-deprived, then incubated for 48 hours in a maintenance mediacontaining 50 μg/ml of sodium heparin and 2 ng/ml TGF-β. Immunoblottingof pooled, precipitated media samples indicated that TGF-β markedlyincreased the secretion of full-length (36-39 kDa) CTGF as demonstratedin FIG. 6A. However, even more pronounced was the induction of themolecule(s) appearing at 18-20 kDa. This smaller moiety corresponds insize to half of the full-length CTGF molecule. Quantitative analysis byELISA of the individual samples, prior to being pooled and precipitatedfor immunoblotting, demonstrated a 2.5-fold enhancement of totalsecreted CTGF in response to TGF-β treatment (see FIG. 6B).

Example 4

Mesangial Cell Expression Of CTGF: Regulation By Glucose Concentrations.To determine if CTGF expression might also be altered by the ambientconcentration of glucose, mesangial cell cultures continuously grown in5 mM glucose were incubated for 14 days in growth media containing 35 mMglucose. The time was chosen because previous studies demonstrated thatthis period was required for the full induction of ECM proteinproduction. (See, e.g., Pugliese et al., 1997, J Am Soc Nephrol8(3):406-414.) As shown in FIG. 7, mesangial cells grown in mediumcontaining 5 mM glucose concentration demonstrated minimal levels ofCTGF message. However, following long-term exposure to an increasedglucose concentration, mesangial cell transcripts for CTGF were markedlyupregulated, reaching a 7-fold level above control, as determined byquantitative image analysis as depicted in FIG. 7.

To examine the effects of high glucose exposure on the secretion of CTGFprotein, serum-deprived cultures that shortened the exposure time to 48hours, and included sodium heparin in the medium were used. Thisprotocol allowed a comparison to the effects of TGF-β. Immunoblotting ofpooled and precipitated media samples indicated that exposure to 20 mMof glucose increased the amount of CTGF secreted as shown in FIG. 6A.Interestingly, however, this stimulation appeared to be limited to thefull-length molecule only. Quantitation of secreted CTGF protein byELISA, prior to pooling and precipitation demonstrated a 2-foldinduction by high extracellular glucose levels as demonstrated in FIG.6B, which is an increase similar to that induced by TGF-β, under theexperimental conditions selected. To determine if the observed increasein CTGF could be due to an osmolar effect, the experiments were repeatedusing a mannitol. Under these conditions, there was no induction of CTGFreleased as measured by ELISA (5 mM glucose, 2.39±0.28 ng/10⁶ cells; 5mM glucose plus 15 mM mannitol, 1.94±0.32), and no change in thedistribution of CTGF forms secreted as determined by immunoblotting.

Example 5

TGF-β Block Of High Glucose-Induced CTGF Production. To determine ifTGF-β is responsible for CTGF production by mesangial cells in thepresence of high glucose, mesangial cells were cultured for 14 days inthe presence of either 5 mM glucose or 20 mM glucose, and were seededand grown under the same glucose conditions for an additional 8 dayperiod. On day 4, the cultures were serum-deprived, and half received 20μg/ml of an antibody that neutralizes TGF-β1, 2 and 3 activity. Freshantibody was added daily, and the media was replaced 24 hours prior tocollection. Measurement of CTGF secretion by ELISA demonstrated astimulatory effect of high glucose as depicted in FIG. 8. However,neutralization of TGF-β activity in these cultures blocked the inductionof CTGF by high glucose. While the constitutive secretion of CTGF in thepresence of normal concentrations of glucose also appeared somewhatreduced by the presence of a TGF-β antibody, this change was notstatistically significant (p=0.09). Also non-significant (p=0.075) wasthe difference in CTGF levels in normal glucose- and highglucose-treated cells when TGF-β was neutralized (see FIG. 8).

Example 6

Glucose transporter expression and CTGF production. Mesangial cellstransduced with the human glucose transporter 1 (GLUT1) gene producing aline designated, MCGT1, demonstrated a 10-fold increase in GLUT1protein, a 5-fold increase in glucose uptake and a 2-3 fold increase inthe synthesis of collagen types I and IV, fibronectin and laminin, ascompared to a control mesangial cell line, designated MCLacZ, andtransduced with the bacterial P-galactosidase gene. These cell lineswere used to demonstrate that the increase in intracellular glucose,rather than simply the extracellular glucose concentration per se, isthe major determinant of exaggerated extracellular matrix formation bymesangial cells in culture. (See, e.g., Helig et al., 1995, supra.) Todetermine if CTGF is also increased in this in vitro model of diabetes,MCCT1 and MCLacZ cells were seeded and grown for 48 hours in RPMI with20% NuSerum, 8 mM glucose. Cells were then washed twice in serum-freemedia and fresh RPMI containing 1% FCS added. Conditioned media werecollected 24 hours later and CTGF protein levels were determined byELISA. Approximately 80 ng of CTGF/10⁶ cells was detected in the mediaof the control MCLacZ cultures whereas the level nearly doubled (147ng/10⁶ cells) in MCGT1 cultures as shown in FIG. 9.

Example 7

Mesangial Cell Expression Of CTGF: Regulation By Cyclic MechanicalStrain. To determine if cyclic mechanical strain was also a factorcapable of altering mesangial cell expression of CTGF, cells were seededinto collagen-coated flexible-bottom plates, then after overnightincubation, and subjected to either stretch or maintained under staticconditions. Stretching was set at 3 cycles per minute and 19% maximumelongation using the computer-controlled system previously described inRiser et al., 1992, supra, and Riser et al., 1996, supra. This degree ofstretching was chosen to approximate the mechanical force experienced bymesangial cells in vivo. (See, e.g., Cartes et al., 1997, supra.)

At the designated periods, the cells were lysed and total RNAs extractedand probed for CTGF transcripts. Cyclic stretching induced a rapid andmarked increase in CTGF message as shown in FIG. 9. Quantitative imageanalysis of the Northern blot showed that levels of CTGF mRNA increasedmore than 2-fold by 4 hours and remained elevated at this level after 8hours of stretching. Additional experiments demonstrated that CTGFtranscripts were significantly increased even after 48 hours of stretch.

Example 8

Blockade Of Stimulated Collagen Production By Anti-CTGF Antibody.

Mesangial cells were grown for 4 days in RPMI medium with 20% NU-SERUMmedia supplement (Collaborative Research), the medium was replaced withone containing 1% FCS (serum-deprived conditions) and 0 or 5 ng/ml ofTGF-β2. Half of the cultures received anti-CTGF antibody (goat affinitypurified, pGAP) and the other half received non-immune goat IgG. Freshantibody was added daily, and the media was replaced 24 hours prior tocollection. Media from individual wells were tested by ELISA. As shownin FIG. 11, treatment with anti-CTGF antibody did not alter theproduction of baseline collagen, but completely blocked the increasedproduction due to TGF-β. There was no significant difference between theamount of collagen produced in unstimulated cultures as compared to thatof cultures stimulated by TGF-β, but treated by anti-CTGF antibody.

Example 9

Blockade Of Stimulated Mesangial Cell Proliferation by Anti-CTGFAntibody. Mesangial cells were grown for four days in RPMI medium with20% NU-SERUM media supplement (Collaborative Research), the medium wasreplaced with one containing 1% FCS (serum-deprived conditions) and 0 or5 ng/ml of TGF-β2. Half of the cultures received anti-CTGF antibody(goat affinity purified, pGAP) and the other half non-immune goat IgG.Fresh antibody was added daily, and the media was replaced 24 hoursprior to collection. Media from individual wells were tested by ELISA.As shown in FIG. 12, TGF-β treatment significantly induced (87%)mesangial cell proliferation. Treatment with anti-CTGF antibodysignificantly reduced (approximately 50%), the induction of cellproliferation. The same antibody treatment had no effect on basalproliferation, i.e., under unstimulated conditions.

Example 10

CTGF Expression In Experimental Diabetic Nephropathy. To determine ifCTGF is upregulated in early diabetic nephropathy, studies were carriedout on diabetic db/db mice, and the results compared to those fromage-matched nondiabetic db/m littermates. At 5 months of age,approximately 3.5 months after the onset of diabetes, animals wereevaluated for blood glucose levels, total weight, proteinuria, andmesangial expansion. At the time of sacrifice, mean blood glucoselevels, as well as body weights, were significantly greater in the db/dbanimals as shown in the following Table 1. TABLE 1 Control db/m Diabeticdb/db Blood Glucose   142 ± 19.0 mg/dL, n = 8   485 ± 58.0, n = 6 P <0.001 Weight  29.2 ± 1.00 g, n = 8  43.1 ± 7.30, n = 6 P < 0.001Proteinuria  2.32 ± 1.07 mg/24 h, n = 10  2.78 ± 0.93, n = 9 P = 0.330Glomerular Sclerosis 0.101 ± 0.048, n = 17 0.649 ± 0.369, n = 6 P =0.003

As shown in FIG. 13, inspection of the renal tissue by light microscopydemonstrated that the diabetic animals exhibited noticeable, butminimal, glomerular changes consistent with early diabeticglomerulosclerosis, i.e. mild mesangial matrix expansion withoutapparent tubulointerstitial disease. In addition, Table 1 shows that thelevel of proteinuria was not significantly greater than in controls.Semiquantitative analysis of the glomerular changes demonstrated thatthe observed mesangial expansion in the diabetic animals was indeedconsistent, but of mild intensity. A value of zero (0) represents nolesion and a value of one (1) represents minimal mesangial expansion inthe majority of glomeruli, without basement membrane thickening.

Northern analysis of whole kidney RNAs indicated that the CTGF messagelevels were markedly increased in 4 out of 5 diabetic mice as shown inFIG. 14A. These changes were mirrored by parallel changes in fibronectintranscript levels. Quantitation of results yielded a mean 103% increasein CTGF expression while fibronectin levels were 80% greater than in thecontrols as shown in FIG. 14B and FIG. 14C. Moreover, transcript levelswere detected in competitive RT-PCR for CTGF mRNA in a single samplefrom diabetic mouse glomeruli as compared to the control GAPDH sample asdepicted in FIG. 15A and FIG. 15B. Analysis of microdissected glomeruliidentified multiple animals (5 diabetic and 5 control groups), that by acompetitive and quantitative RT-PCR method (described above), identifieda low, but measurable, transcript level of CTGF in the glomeruli ofcontrol animals. (See FIG. 16.) In mice with diabetes, the level of CTGFwas dramatically increased by 27-fold. (See FIG. 16.) The upregulationof glomerular CTGF mRNA was accompanied by a nearly 5-fold increase inthe amount of fibronectin mRNA. These large differences were not due todissimilar glomerular size resulting from diabetic hypertrophy, sincethe level of GAPDH message was not significantly increased in diabeticanimals as compared to controls (control, 1.39±0.524×10⁻¹attomoles/glomerulus; diabetic 2.59±0.307; P>0.05). Therefore, adramatic increase in CTGF expression was documented at a time whenchanges in the kidney were minimal.

C. Detection of CTGF in Samples as an Indicator of Renal DiabetesAssociated Disorders

Example 11

The Presence And Stability Of Urinary CTGF. To examine whether CTGFprotein was secreted in the urine, and to examine the stability of theCTGF molecule after its secretion in the urine, samples of urine werecollected from a healthy donor and divided into five, 25-ml aliquots.Various amounts of rhCTGF, ranging from 25 to 750 ng, were added to fourof the five aliquots (the “spiked samples”). The fifth aliquot served asa control, receiving no added CTGF (the “unspiked sample). All sampleswere frozen, and stored at −70° C., then later thawed and clarified bycentrifugation. Following heparin sepharose quantitative extraction ofsamples, immunoblotting was performed using CTGF specific antibody asindicated in FIG. 17. The results identified the presence of a scarcelydetectable level of CTGF secretion in the urine of the unspiked sample.Further, the stability of the CTGF protein in the urine was demonstratedby the progressive increase in CTGF recovered in the spiked samples.Comparison of sample lanes with that containing freshly added rhCTGF (35ng) indicated that CTGF was largely, if not entirely, preserved.

Example 12

CTGF In The Urine Of Renal Patients. The quantity and/or molecular formof CTGF present in the urine that might be altered in patients withestablished nephropathy, including that associated with diabetes, wasinvestigated. Urine samples from 8 ambulatory patients being treated fora variety of kidney diseases, of which 3 had a history of diabetes, werecollected and frozen during routine visits (Nephrology and HypertensionClinic, Henry Ford Hospital). Similarly, samples were also obtained from3 normal healthy volunteers with no history of kidney disease. Allsamples were later batch-thawed and processed above. CTGF was detectedin 1 of 3 normal volunteers, and in all patient samples as shown in FIG.18. Immunoreactive CTGF appeared in 3 different molecular forms. A CTGFband (doublet) was present in 1 control sample and 4 of 8 patientssamples. Interestingly, a large molecular weight band, approximately 200kDA, was present in every patient sample, appearing only as a very faintband in a single control sample. This large band likely represents CTGFin complex with a second, unknown urinary protein. Even more intriguingwas a unique small CTGF fragment, approximately 9-12 kDa. This smallermoiety appears to be equivalent to the heparin binding C-terminalquarter fragment of CTGF, and was present in the urine of all 3 diabeticpatients, but not present in nondiabetic patients or healthy controls.Interestingly, this product may compare to the CTGF fragment produced bymesangial cells in culture when stimulated by high glucoseconcentrations of TGF-β.

In a separate experiment, CTGF in human urine samples was measure byELISA, and presented in the following Table 2. TABLE 2 PatientPopulation Patient Healthy Kidney Diabetic: No Number Control DiseaseKidney Disease 1 0.81 0.36 0.68 2 0.43 1.22 7.12 3 0.84 5.46 5.68 4 0.672.53 0.40 5 5.72 0.56 6 0.78 3.84 7 3.47 Mean CTGF 0.69 2.79 3.05 ±SE0.10 0.83 1.12

Table 2 compares the amounts of CTGF detected in healthy volunteers topatients with kidney disease (in some cases associated with diabetes),or to patients with 5 to 10 years with diabetes, but without kidneydisease. Each group had 4 to 7 samples from different individuals.Amounts of CTGF/ml were first determined by ELISA, comparing samplevalues to a standard curve using serial dilutions of a known quantity ofrhCTGF. To standardize results (i.e. to account for variation in theproduction of urine), amounts of CTGF (CTGF/ml) were then divided by theurine creatinine from the same patient, determined from the same urine.

The results demonstrate that healthy individuals demonstrateconsistently low levels of urinary CTGF. However, among those withkidney disease the mean level of CTGF increased 4-fold. In thosepatients with diabetes, but as yet undiagnosed kidney disease, there wasa similar 4.4-fold increase. It was expected that because onlyapproximately 40% of those with diabetes will go on to developnephropathy, a similar percent of patients would exhibit increased CTGFlevels. Interestingly, of the 6 diabetic patients tested, 3, or 50%,demonstrated clearly elevated CTGF levels. The remaining patientsappeared to have values similar to those of the healthy volunteers.

Various modifications and variations of the described methods andsystems of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims. All patents, publications, and other references cited herein areincorporated by reference herein in their entirety.

1. A method of identifying an agent that modulates CTGF induction byglucose, the method comprising (a) culturing a first cell population inmedia containing high glucose; (b) culturing a second cell population inmedia containing high glucose and an agent; (c) measuring CTGF level inthe culture media from the first and second cell populations; and (d)comparing CTGF level in the culture media from the second cellpopulation to CTGF level in the culture media from the first cellpopulation, wherein a difference in CTGF level between the second andfirst cell populations is indicative of an agent that modulates CTGFinduction by glucose.
 2. The method of claim 1, wherein the glucoseconcentration in the media containing high glucose is at least about 20mM.
 3. The method of claim 1, wherein the glucose concentration in themedia containing high glucose is at least about 35 mM.
 4. The method ofclaim 1, wherein the first and second cell populations are of the samecell type and are fibroblasts.
 5. The method of claim 4, wherein thefibroblasts are kidney mesangial cells.
 6. An agent that modulates CTGFinduction by glucose, wherein the agent is identified by the method ofclaim
 1. 7. The agent of claim 6, wherein the agent additionallyincreases cyclic nucleotide level in a cell.
 8. A method of reducingexpression of CTGF in a subject having elevated blood glucose, themethod comprising administering the agent of claim 6 to a subject havingor at risk for having high blood glucose.
 9. A method of identifying anagent that modulates CTGF induction associated with mechanical stress,the method comprising (a) culturing a first cell population in theabsence of agent under conditions that place the cells under increasedmechanical stress; (b) culturing a second cell population in thepresence of agent under conditions that place the cells under increasedmechanical stress; (c) measuring CTGF level in the culture media fromfirst and second cell populations; and (d) comparing CTGF level in theculture media from the second cell population to CTGF level in theculture media from the first cell population, wherein a difference inCTGF level between the second and first cell populations is indicativeof an agent that modulates CTGF induction associated with mechanicalstress.
 10. The method of claim 9, wherein mechanical stress is cyclicstretching of cells up to and including 19% maximum elongation.
 11. Themethod of claim 9, wherein the first and second cell populations are ofthe same cell type and are fibroblasts.
 12. The method of claim 11,wherein the fibroblasts are kidney mesangial cells.
 13. An agent thatmodulates CTGF induction associated with mechanical stress, wherein theagent is identified by the method of claim
 9. 14. A method of reducingexpression of CTGF in a subject having elevated blood pressure, themethod comprising administering the agent of claim 13 to a subjecthaving elevated blood pressure.
 15. A method of treating or delayingonset of a disorder associated with elevated blood glucose, the methodcomprising administering to a subject in need an effective amount of anagent that modulates, regulates, or inhibits the expression or activityof CTGF or fragments thereof.
 16. The method of claim 15, wherein thesubject has or is at risk of having hyperglycemia.
 17. The method ofclaim 15, wherein the agent is an antibody that specifically binds toCTGF or fragments thereof.
 18. The method of claim 15, wherein the agentis an antisense oligonucleotide that specifically binds to apolynucleotide sequence encoding CTGF.
 19. The method of claim 15,wherein the agent is a small molecule.
 20. The method of claim 19,wherein the agent additionally increases cyclic nucleotide levels in acell.
 21. The method of claim 15, wherein the disorder is diabetes. 22.The method of claim 15, wherein the disorder is a renal disorder. 23.The method of claim 22, wherein the renal disorder is selected from thegroup consisting of glomerulosclerosis, glomerulonephritis, and diabeticnephropathy.
 24. The method of claim 15, wherein the disorder is anocular disorder.
 25. The method of claim 24, wherein the ocular disorderis diabetic retinopathy.
 26. A method of treating or delaying onset of adisorder associated with elevated blood pressure, the method comprisingadministering to a subject in need an effective amount of an agent thatmodulates, regulates, or inhibits the expression or activity of CTGF orfragments thereof.
 27. The method of claim 26, wherein the agent is anantibody that specifically binds to CTGF or fragments thereof.
 28. Themethod of claim 26, wherein the agent is an antisense oligonucleotidethat specifically binds to a polynucleotide sequence encoding CTGF. 29.The method of claim 26, wherein the agent is a small molecule.
 30. Themethod of claim 29, wherein the agent additionally increases cyclicnucleotide levels in a cell.
 31. The method of claim 26, wherein thedisorder is diabetes.
 32. The method of claim 26, wherein the disorderis a renal disorder.
 33. The method of claim 32, wherein the renaldisorder is selected from glomerulosclerosis, glomerulonephritis, anddiabetic nephropathy.
 34. The method of claim 26, wherein the disorderis an ocular disorder.
 35. The method of claim 34, wherein the oculardisorder is diabetic retinopathy.
 36. A method of treating or delayingonset of a disorder associated with renal hypertension, the methodcomprising administering to a subject in need an effective amount of anagent that modulates, regulates, or inhibits the expression or activityof CTGF or fragments thereof.
 37. The method of claim 36, wherein theagent is an antibody that specifically binds to CTGF or fragmentsthereof.
 38. The method of claim 36, wherein the agent is an antisenseoligonucleotide that specifically binds to a polynucleotide sequenceencoding CTGF.
 39. The method of claim 36, wherein the agent is a smallmolecule.
 40. The method of claim 39, wherein the agent additionallyincreases cyclic nucleotide levels in a cell.
 41. The method of claim36, wherein the disorder is diabetes.
 42. The method of claim 36,wherein the disorder is a renal disorder.
 43. The method of claim 42,wherein the renal disorder is selected from glomerulosclerosis,glomerulonephritis, and diabetic nephropathy.